Apolipoprotein A-I agonists and their use to treat dyslipidemic disorders

ABSTRACT

The present invention provides peptides and peptide analogues that mimic the structural and pharmacological properties of human ApoA-I. The peptides and peptide analogues are useful to treat a variety of disorders associated with dyslipidemia.

1. INTRODUCTION

[0001] The invention relates to apolipoprotein A-I (ApoA-I) agonistcompositions for treating disorders associated with dyslipoproteinemia,including hypercholesterolemia, cardiovascular disease, atherosclerosis,restenosis, and other disorders such as septic shock.

2. BACKGROUND OF THE INVENTION

[0002] Circulating cholesterol is carried by plasmalipoproteins—particles of complex lipid and protein composition thattransport lipids in the blood. Low density lipoproteins (LDL), and highdensity lipoproteins (HDL) are the major cholesterol carriers. LDL arebelieved to be responsible for the delivery of cholesterol from theliver (where it is synthesized or obtained from dietary sources) toextrahepatic tissues in the body. The term “reverse cholesteroltransport” describes the transport of cholesterol from extrahepatictissues to the liver where it is catabolized and eliminated. It isbelieved that plasma HDL particles play a major role in the reversetransport process, acting as scavengers of tissue cholesterol.

[0003] The evidence linking elevated serum cholesterol to coronary heartdisease is overwhelming. For example, atherosclerosis is a slowlyprogressive disease characterized by the accumulation of cholesterolwithin the arterial wall. Compelling evidence supports the concept thatlipids deposited in atherosclerotic lesions are derived primarily fromplasma LDL; thus, LDLs have popularly become known as the “bad”cholesterol. In contrast, HDL serum levels correlate inversely withcoronary heart disease—indeed, high serum levels of HDL are regarded asa negative risk factor. It is hypothesized that high levels of plasmaHDL are not only protective against coronary artery disease, but mayactually induce regression of atherosclerotic plaques (e.g., see Badimonet al., 1992, Circulation 86 (Suppl. III):86-94). Thus, HDL havepopularly become known as the “good” cholesterol.

2.1. Cholesterol Transport

[0004] The fat-transport system can be divided into two pathways: anexogenous one for cholesterol and triglycerides absorbed from theintestine, and an endogenous one for cholesterol and triglyceridesentering the bloodstream from the liver and other non-hepatic tissue.

[0005] In the exogenous pathway, dietary fats are packaged intolipoprotein particles called chylomicrons which enter the bloodstreamand deliver their triglycerides to adipose tissue (for storage) and tomuscle (for oxidation to supply energy). The remnant of the chylomicron,containing cholesteryl esters, is removed from the circulation by aspecific receptor found only on liver cells. This cholesterol thenbecomes available again for cellular metabolism or for recycling toextrahepatic tissues as plasma lipoproteins.

[0006] In the endogenous pathway, the liver secretes a large,very-low-density lipoprotein particle (VLDL) into the bloodstream. Thecore of VLDLs consists mostly of triglycerides synthesized in the liver,with a smaller amount of cholesteryl esters (either synthesized in theliver or recycled from chylomicrons). Two predominant proteins aredisplayed on the surface of VLDLs, apoprotein B-100 and apoprotein E.When a VLDL reaches the capillaries of adipose tissue or of muscle, itstriglycerides are extracted resulting in a new kind of particle,decreased in size and enriched in cholesteryl esters but retaining itstwo apoproteins, called intermediate-density lipoprotein (IDL).

[0007] In human beings, about half of the IDL particles are removed fromthe circulation quickly (within two to six hours of their formation),because they bind tightly to liver cells which extract their cholesterolto make new VLDL and bile acids. The IDL particles which are not takenup by the liver remain in the circulation longer. In time, theapoprotein E dissociates from the circulating particles, converting themto LDL having apoprotein B-100 as their sole protein.

[0008] Primarily, the liver takes up and degrades most of thecholesterol to bile acids, which are the end products of cholesterolmetabolism. The uptake of cholesterol containing particles is mediatedby LDL receptors, which are present in high concentrations onhepatocytes. The LDL receptor binds both apoprotein E and apoproteinB-100, and is responsible for binding and removing both IDLs and LDLsfrom the circulation. However, the affinity of apoprotein E for the LDLreceptor is greater than that of apoprotein B-100. As a result, the LDLparticles have a much longer circulating life span than IDLparticles—LDLs circulate for an average of two and a half days beforebinding to the LDL receptors in the liver and other tissues. High serumlevels of LDL (the “bad” cholesterol) are positively associated withcoronary heart disease. For example, in atherosclerosis, cholesterolderived from circulating LDLs accumulates in the walls of arteriesleading to the formation of bulky plaques that inhibit the flow of blooduntil a clot eventually forms, obstructing the artery causing a heartattack or stroke.

[0009] Ultimately, the amount of intracellular cholesterol liberatedfrom the LDLs controls cellular cholesterol metabolism. The accumulationof cellular cholesterol derived from VLDLs and LDLs controls threeprocesses: first, it reduces cellular cholesterol synthesis by turningoff the synthesis of HMGCOA reductase—a key enzyme in the cholesterolbiosynthetic pathway. Second, the incoming LDL-derived cholesterolpromotes storage of cholesterol by activating ACAT—the cellular enzymewhich converts cholesterol into cholesteryl esters that are deposited instorage droplets. Third, the accumulation of cholesterol within the celldrives a feedback mechanism that inhibits cellular synthesis of new LDLreceptors. Cells, therefore, adjust their complement of LDL receptors sothat enough cholesterol is brought in to meet their metabolic needs,without overloading. (For a review, see Brown & Goldstein, In, ThePharmacological Basis Of Therapeutics, 8th Ed., Goodman & Gilman,Pergamon Press, NY, 1990, Ch. 36, pp. 874-896).

2.2. Reverse Cholesterol Transport

[0010] In sum, peripheral (non-hepatic) cells obtain their cholesterolfrom a combination of local synthesis and the uptake of preformed sterolfrom VLDLs and LDLs. In contrast, reverse cholesterol transport (RCT) isthe pathway by which peripheral cell cholesterol can be returned to theliver for recycling to extrahepatic tissues, or excretion into theintestine in bile, either in modified or in oxidized form as bile acids.The RCT pathway represents the only means of eliminating cholesterolfrom most extrahepatic tissues, and is crucial to maintenance of thestructure and function of most cells in the body.

[0011] The RCT consists mainly of three steps: (a) cholesterol efflux,the initial removal of cholesterol from various pools of peripheralcells; (b) cholesterol esterification by the action oflecithin:cholesterol acyltransferase (LCAT), preventing a re-entry ofeffluxed cholesterol into cells; and (c) uptake/delivery of HDLcholesteryl ester to liver cells. The RCT pathway is mediated by HDLs.HDL is a generic term for lipoprotein particles which are characterizedby their high density. The main lipidic constituents of HDL complexesare various phospholipids, cholesterol (ester) and triglycerides. Themost prominent apolipoprotein components are A-I and A-II whichdetermine the functional characteristics of HDL; furthermore minoramounts of apolipoprotein C-I, C-II, C-III, D, E, J, etc. have beenobserved. HDL can exist in a wide variety of different sizes anddifferent mixtures of the above-mentioned constituents depending on thestatus of remodeling during the metabolic RCT cascade.

[0012] The key enzyme involved in the RCT pathway is LCAT. LCAT isproduced mainly in the liver and circulates in plasma associated withthe HDL fraction. LCAT converts cell derived cholesterol to cholesterylesters which are sequestered in HDL destined for removal. Cholesterylester transfer protein (CETP) and phospholipid transfer protein (PLTP)contribute to further remodeling the circulating HDL population. CETPcan move cholesteryl esters made by LCAT to other lipoproteins,particularly ApoB-containing lipoproteins, such as VLDL and LDL. PLTPsupplies lecithin to HDL. HDL triglycerides can be catabolized by theextracellular hepatic triglyceride lipase, and lipoprotein cholesterolis removed by the liver via several mechanisms.

[0013] Each HDL particle contains at least one copy (and usually two tofour copies) of ApoA-I. ApoA-I is synthesized by the liver and smallintestine as preproapolipoprotein which is secreted as a proprotein thatis rapidly cleaved to generate a mature polypeptide having 243 aminoacid residues. ApoA-I consists mainly of 6 to 8 different 22 amino acidrepeats spaced by a linker moiety which is often proline, and in somecases consists of a stretch made up of several residues. ApoA-I formsthree types of stable complexes with lipids: small, lipid-poor complexesreferred to as pre-beta-1 HDL; flattened discoidal particles containingpolar lipids (phospholipid and cholesterol) referred to as pre-beta-2HDL; and spherical particles containing both polar and nonpolar lipids,referred to as spherical or mature HDL (HDL₃ and HDL₂). Most HDL in thecirculating population contain both ApoA-I and ApoA-II (the second majorHDL protein) and are referred to herein as the AI/AII-HDL fraction ofHDL. However, the fraction of HDL containing only ApoA-I (referred toherein as the AI-HDL fraction) appear to be more effective in RCT.Certain epidemiologic studies support the hypothesis that the AI-HDLfraction is anti-atherogenic. (Parra et al., 1992, Arterioscler. Thromb.12:701-707; Decossin et al., 1997, Eur. J. Clin. Invest. 27:299-307).

[0014] Although the mechanism for cholesterol transfer from the cellsurface (i.e., cholesterol efflux) is unknown, it is believed that thelipid-poor complex, pre-beta-1 HDL is the preferred acceptor forcholesterol transferred from peripheral tissue involved in RCT. (SeeDavidson et al., 1994, J. Biol. Chem. 269:22975-22982; Bielicki et al.,1992, J. Lipid Res. 33:1699-1709; Rothblat et al., 1992, J. Lipid Res.33:1091-1097; and Kawano et al., 1993, Biochemistry 32:5025-5028; Kawanoet al., 1997, Biochemistry 36:9816-9825). During this process ofcholesterol recruitment from the cell surface, pre-beta-1 HDL is rapidlyconverted to pre-beta-2 HDL. PLTP may increase the rate of pre-beta-2disc formation, but data indicating a role for PLTP in RCT is lacking.LCAT reacts preferentially with discoidal and spherical HDL,transferring the 2-acyl group of lecithin or other phospholipids to thefree hydroxyl residue of cholesterol to generate cholesteryl esters(retained in the HDL) and lysolecithin. The LCAT reaction requiresApoA-I as activator; i.e., ApoA-I is the natural cofactor for LCAT. Theconversion of cholesterol to its ester sequestered in the HDL preventsre-entry of cholesterol into the cell, the result being that cholesterylesters are destined for removal. Cholesteryl esters in the mature HDLparticles in the AI-HDL fraction (i.e., containing ApoA-I and noApoA-II) are removed by the liver and processed into bile moreeffectively than those derived from HDL containing both ApoA-I andApoA-II (the AI/AII-HDL fraction). This may be due, in part, to the moreeffective binding of AI-HDL to the hepatocyte membrane. The existence ofan HDL receptor has been hypothesized, and recently a scavengerreceptor, SR-BI, was identified as an HDL receptor (Acton et al., 1996,Science 271:518-520; Xu et al., 1997, J. Lipid Res. 38:1289-1298). TheSR-BI is expressed most abundantly in steroidogenic tissues (e.g., theadrenals), and in the liver (Landshulz et al., 1996, J. Clin. Invest.98:984-995; Rigotti et al., 1996, J. Biol. Chem. 271:33545-33549).

[0015] CETP does not appear to play a major role in RCT, and instead isinvolved in the metabolism of VLDL-and LDL-derived lipids. However,changes in CETP activity or its acceptors, VLDL and LDL, play a role in“remodeling” the HDL population. For example, in the absence of CETP,the HDLs become enlarged particles which are not cleared. (For reviewson RCT and HDLs, see Fielding & Fielding, 1995, J. Lipid Res.36:211-228; Barrans et al., 1996, Biochem. Biophys. Acta. 1300:73-85;Hirano et al., 1997, Arterioscler. Thromb. Vasc. Biol. 17(6):1053-1059).

2.3. Current Treatments for Dyslipoproteinemias

[0016] A number of treatments are currently available for lowering serumcholesterol and triglycerides (see, e.g., Brown & Goldstein, supra).However, each has its own drawbacks and limitations in terms ofefficacy, side-effects and qualifying patient population.

[0017] Bile-acid-binding resins are a class of drugs that interrupt therecycling of bile acids from the intestine to the liver; e.g.,cholestyramine (Questran Light®, Bristol-Myers Squibb), and colestipolhydrochloride (Colestid®, The Upjohn Company). When taken orally, thesepositively-charged resins bind to the negatively charged bile acids inthe intestine. Because the resins cannot be absorbed from the intestine,they are excreted carrying the bile acids with them. The use of suchresins, however, at best only lowers serum cholesterol levels by about20%, and is associated with gastrointestinal side-effects, includingconstipation and certain vitamin deficiencies. Moreover, since theresins bind other drugs, other oral medications must be taken at leastone hour before or four to six hours subsequent to ingestion of theresin; thus, complicating heart patient's drug regimens.

[0018] The statins are cholesterol lowering agents that blockcholesterol synthesis by inhibiting HMGCOA reductase—the key enzymeinvolved in the cholesterol biosynthetic pathway. The statins, e.g.,lovastatin (Mevacor®, Merck & Co., Inc.), and pravastatin (Pravachol®,Bristol-Myers Squibb Co.) are sometimes used in combination withbile-acid-binding resins. The statins significantly reduce serumcholesterol and LDL-serum levels, and slow progression of coronaryatherosclerosis. However, serum HDL cholesterol levels are onlymoderately increased. The mechanism of the LDL lowering effect mayinvolve both reduction of VLDL concentration and induction of cellularexpression of LDL-receptor, leading to reduced production and/orincreased catabolism of LDLs. Side effects, including liver and kidneydysfunction are associated with the use of these drugs (Physicians DeskReference, Medical Economics Co., Inc., Montvale, N.J., 1997). Recently,the FDA has approved atorvastatin (an HMGCOA reductase inhibitordeveloped by Parke-Davis) (Warner Lambert) for the market to treat rarebut urgent cases of familial hypercholesterolemia (1995, Scrip20(19):10).

[0019] Niacin, or nicotinic acid, is a water soluble vitamin B-complexused as a dietary supplement and antihyperlipidemic agent. Niacindiminishes production of VLDL and is effective at lowering LDL. In somecases, it is used in combination with bile-acid binding resins. Niacincan increase HDL when used at adequate doses, however, its usefulness islimited by serious side effects when used at such high doses.

[0020] Fibrates are a class of lipid-lowering drugs used to treatvarious forms of hyperlipidemia (i.e., elevated serum triglycerides)which may also be associated with hypercholesterolemia. Fibrates appearto reduce the VLDL fraction and modestly increase HDL—however theeffects of these drugs on serum cholesterol is variable. In the UnitedStates, fibrates have been approved for use as antilipidemic drugs, buthave not received approval as hypercholesterolemia agents. For example,clofibrate (Atromid-S®, Wyeth-Ayerst Laboratories) is an antilipidemicagent which acts (via an unknown mechanism) to lower serum triglyceridesby reducing the VLDL fraction. Although serum cholesterol may be reducedin certain patient subpopulations, the biochemical response to the drugis variable, and is not always possible to predict which patients willobtain favorable results. Atromid-S® has not been shown to be effectivefor prevention of coronary heart disease. The chemically andpharmacologically related drug, gemfibrozil (Lopid®, Parke-Davis) is alipid regulating agent which moderately decreases serum triglyceridesand VLDL cholesterol, and moderately increases HDL cholesterol—the HDL₂and HDL₃ subfractions as well as both ApoA-I and A-II (i.e., theAI/AII-HDL fraction). However, the lipid response is heterogeneous,especially among different patient populations. Moreover, whileprevention of coronary heart disease was observed in male patientsbetween 40-55 without history or symptoms of existing coronary heartdisease, it is not clear to what extent these findings can beextrapolated to other patient populations (e.g., women, older andyounger males). Indeed, no efficacy was observed in patients withestablished coronary heart disease. Serious side-effects are associatedwith the use of fibrates including toxicity such as malignancy,(especially gastrointestinal cancer), gallbladder disease and anincreased incidence in non-coronary mortality. These drugs are notindicated for the treatment of patients with high LDL or low HDL astheir only lipid abnormality (Physician's Desk Reference, 1997, MedicalEconomics Co., Inc. Montvale, N.J.).

[0021] Oral estrogen replacement therapy may be considered for moderatehypercholesterolemia in post-menopausal women. However, increases in HDLmay be accompanied with an increase in triglycerides. Estrogen treatmentis, of course, limited to a specific patient population (postmenopausalwomen) and is associated with serious side effects including inductionof malignant neoplasms, gall bladder disease, thromboembolic disease,hepatic adenoma, elevated blood pressure, glucose intolerance, andhypercalcemia.

[0022] Thus, there is a need to develop safer drugs that are efficaciousin lowering serum cholesterol, increasing HDL serum levels, preventingcoronary heart disease, and/or treating existing disease, especiallyatherosclerosis.

2.4. ApoA-I as a Target

[0023] None of the currently available drugs for lowering cholesterolsafely elevate HDL levels and stimulate RCT—most appear to operate onthe cholesterol transport pathway, modulating dietary intake, recycling,synthesis of cholesterol, and the VLDL population.

[0024] While it is desirable to find drugs that stimulate cholesterolefflux and removal, several potential targets in the RCT exist—e.g.,LCAT, HDL and its various components (ApoA-I, ApoA-II andphospholipids), PLTP, and CETP—and it is not known which target would bemost effective at achieving desirable lipoprotein profiles andprotective effects. Perturbation of any single component in the RCTpathway ultimately affects the composition of circulating lipoproteinpopulations, and the efficiency of RCT.

[0025] Several lines of evidence based on data obtained in vivoimplicate the HDL and its major protein component, ApoA-I, in theprevention of atherosclerotic lesions, and potentially, the regressionof plaques—making these attractive targets for therapeutic intervention.First, an inverse correlation exists between serum ApoA-I (HDL)concentration and atherogenesis in man (Gordon & Rifkind, 1989, N. Eng.J. Med. 321:1311-1316; Gordon et al., 1989, Circulation 79:8-15).Indeed, specific subpopulations of HDL have been associated with areduced risk for atherosclerosis in humans (Miller, 1987, Amer. Heart113:589-597; Cheung et al., 1991, Lipid Res. 32:383-394); Fruchart &Ailhaud, 1992, Clin. Chem. 38:79).

[0026] Second, animal studies support the protective role of ApoA-I(HDL). Treatment of cholesterol fed rabbits with ApoA-I or HDL reducedthe development and progression of plaque (fatty streaks) incholesterol-fed rabbits. (Koizumi et al., 1988, J. Lipid Res.29:1405-1415; Badimon et al., 1989, Lab. Invest. 60:455-461; Badimon etal., 1990, J. Clin. Invest. 85:1234-1241). However, the efficacy varieddepending upon the source of HDL (Beitz et al., 1992, Prostaglandins,Leukotrienes and Essential Fatty Acids 47:149-152; Mezdour et al., 1995,Atherosclerosis 113:237-246).

[0027] Third, direct evidence for the role of ApoA-I was obtained fromexperiments involving transgenic animals. The expression of the humangene for ApoA-I transferred to mice genetically predisposed todiet-induced atherosclerosis protected against the development of aorticlesions (Rubin et al., 1991, Nature 353:265-267). The ApoA-I transgenewas also shown to suppress atherosclerosis in ApoE-deficient mice and inApo(a) transgenic mice (Paszty et al., 1994, J. Clin. Invest.94:899-903; Plump et al., 1994, Proc. Natl. Acad. Sci. USA 91:9607-9611;Liu et al., 1994, J. Lipid Res. 35:2263-2266). Similar results wereobserved in transgenic rabbits expressing human ApoA-I (Duverger, 1996,Circulation 94:713-717; Duverger et al., 1996, Arterioscler. Thromb.Vasc. Biol. 16:1424-1429), and in transgenic rats where elevated levelsof human ApoA-I protected against atherosclerosis and inhibitedrestenosis following balloon angioplasty (Burkey et al., 1992,Circulation, Supplement I, 86:I-472, Abstract No. 1876; Burkey et al.,1995, J. Lipid Res. 36:1463-1473).

[0028] The AI-HDL appear to be more efficient at RCT than the AI/AII-HDLfraction. Studies with mice transgenic for human ApoA-I or Apo-I andApoA-II (AI/AII) showed that the protein composition of HDLsignificantly affects its role—AI-HDL is more anti-atherogenic thanAI/AII-HDL (Schultz et al., 1993, Nature 365:762-764). Parallel studiesinvolving transgenic mice expressing the human LCAT gene demonstratethat moderate increases in LCAT activity significantly changelipoprotein cholesterol levels, and that LCAT has a significantpreference for HDL containing ApoA-I (Francone et al., 1995, J. Clinic.Invest. 96:1440-1448; Berard et al., 1997, Nature Medicine3(7):744-749). While these data support a significant role for ApoA-I inactivating LCAT and stimulating RCT, additional studies demonstrate amore complicated scenario: a major component of HDL that modulatesefflux of cell cholesterol is the phospholipids (Fournier et al., 1996,J. Lipid Res. 37:1704-1711).

[0029] In view of the potential role of HDL, i.e., both ApoA-I and itsassociated phospholipid, in the protection against atheroscleroticdisease, human clinical trials utilizing recombinantly produced ApoA-Iwere commenced, discontinued and apparently re-commenced by UCB Belgium(Pharmaprojects, Oct. 27, 1995; IMS R&D Focus, Jun. 30, 1997; DrugStatus Update, 1997, Atherosclerosis 2(6):261-265); see also M. Erikssonat Congress, “The Role of HDL in Disease Prevention,” Nov. 7-9, 1996,Fort Worth; Lacko & Miller, 1997, J. Lip. Res. 38:1267-1273; andWO94/13819) and were commenced and discontinued by Bio-Tech(Pharmaprojects, Apr. 7, 1989). Trials were also attempted using ApoA-Ito treat septic shock (Opal, “Reconstituted HDL as a Treatment Strategyfor Sepsis,” IBC's 7th International Conference on Sepsis, Apr. 28-30,1997, Washington, D.C.; Gouni et al., 1993, J. Lipid Res. 94:139-146;Levine, WO96/04916). However, there are many pitfalls associated withthe production and use of ApoA-I, making it less than ideal as a drug;e.c., ApoA-I is a large protein that is difficult and expensive toproduce; significant manufacturing and reproducibility problems must beovercome with respect to stability during storage, delivery of an activeproduct and half-life in vivo.

[0030] In view of these drawbacks, attempts have been made to preparepeptides that mimic ApoA-I. Since the key activities of ApoA-I have beenattributed to the presence of multiple repeats of a unique secondarystructural feature in the protein—a class A amphipathic α-helix(Segrest, 1974, FEBS Lett. 38:247-253), most efforts to design peptideswhich mimic the activity of ApoA-I have focused on designing peptideswhich form class A-type amphipathic α-helices.

[0031] Class A-type amphipathic α-helices are unique in that positivelycharged amino acid residues are clustered at the hydrophobic-hydrophilicinterface and negatively charged amino acid residues are clustered atthe center of the hydrophilic face. Furthermore, class A α-helicalpeptides have a hydrophobic angle of less than 180° (Segrest et al.,1990, PROTEINS: Structure, Function and Genetics 8:103-117). The initialde novo strategies to design ApoA-I mimics were not based upon theprimary sequences of naturally occurring apolipoproteins, but ratherupon incorporating these unique Class A helix features into thesequences of the peptide analogues, as well as some of the properties ofthe ApoA-I domains (see, e.g., Davidson et al., 1996, Proc. Natl. Acad.Sci. USA 93:13605-13610; Rogers et al., 1997, Biochemistry 36:288-300;Lins et al., 1993, Biochim. Biophys. Acta biomembranes 1151:137-142; Jiand Jonas, 1995, J. Biol. Chem. 270:11290-11297; Collet et al., 1997,Journal of Lipid Research, 38:634-644; Sparrow and Gotto, 1980, Ann.N.Y. Acad. Sci. 348:187-211; Sparrow and Gotto, 1982, CRC Crit. Rev.Biochem. 13:87-107; Sorci-Thomas et al., 1993, J. Biol. Chem.268:21403-21409; Wang et al., 1996, Biochim. Biophys. Acta 174-184;Minnich et al., 1992, J. Biol. Chem. 267:16553-16560; Holvoet et al.,1995, Biochemistry 34:13334-13342; Sorci-Thomas et al., 1997, J. Biol.Chem. 272(11) :7278-7284; and Frank et al., 1997, Biochemistry36:1798-1806).

[0032] In one study, Fukushima et al. synthesized a 22-residue peptidecomposed entirely of Glu, Lys and Leu residues arranged periodically soas to form an amphipathic α-helix with equal hydrophilic and hydrophobicfaces (“ELK peptide”) (Fukushima et al., 1979, J. Amer. Chem. Soc.101(13):3703-3704; Fukushima et al., 1980, J. Biol. Chem.255:10651-10657). The ELK peptide shares 41% sequence homology with the198-219 fragment of ApoA-I. As studied by quantitative ultrafiltration,gel permeation chromatography and circular dichroism, this ELK peptidewas shown to effectively associate with phospholipids and mimic some ofthe physical and chemical properties of ApoA-I (Kaiser et al., 1983,Proc. Natl. Acad. Sci. USA 80:1137-1140; Kaiser et al., 1984, Science223:249-255; Fukushima et al., 1980, supra; Nakagawa et al., 1985, J.Am. Chem. Soc. 107:7087-7092). Yokoyama et al. concluded from suchstudies that the crucial factor for LCAT activation is simply thepresence of a large enough amphipathic structure (Yokoyama et al., 1980,J. Biol. Chem. 255(15):7333-7339). A dimer of this 22-residue peptidewas later found to more closely mimic ApoA-I than the monomer; based onthese results, it was suggested that the 44-mer, which is punctuated inthe middle by a helix breaker (either Gly or Pro), represented theminimal functional domain in ApoA-I (Nakagawa et al., 1985, supra).

[0033] Another study involved model amphipathic peptides called “LAPpeptides” (Pownall et al., 1980, Proc. Natl. Acad. Sci. USA77(6):3154-3158; Sparrow et al., 1981, In: Peptides:Synthesis-Structure-Function, Roch and Gross, Eds., Pierce Chem. Co.,Rockford, Ill., 253-256). Based on lipid binding studies with fragmentsof native apolipoproteins, several LAP peptides were designed, namedLAP-16, LAP-20 and LAP-24 (containing 16, 20 and 24 amino acid residues,respectively). These model amphipathic peptides share no sequencehomology with the apolipoproteins and were designed to have hydrophilicfaces organized in a manner unlike the class A-type amphipathic helicaldomains associated with apolipoproteins (Segrest et al., 1992, J. LipidRes. 33:141-166). From these studies, the authors concluded that aminimal length of 20 residues is necessary to confer lipid-bindingproperties to model amphipathic peptides.

[0034] Studies with mutants of LAP20 containing a proline residue atdifferent positions in the sequence indicated that a direct relationshipexists between lipid binding and LCAT activation, but that the helicalpotential of a peptide alone does not lead to LCAT activation (Ponsin etal., 1986 J. Biol. Chem. 261(20):9202-9205). Moreover, the presence ofthis helix breaker (Pro) close to the middle of the peptide reduced itsaffinity for phospholipid surfaces as well as its ability to activateLCAT. While certain of the LAP peptides were shown to bind phospholipids(Sparrow et al., supra), controversy exists as to the extent to whichLAP peptides are helical in the presence of lipids (Buchko et al., 1996,J. Biol. Chem. 271(6):3039-3045; Zhong et al., 1994, Peptide Research7(2):99-106).

[0035] Segrest et al. have synthesized peptides composed of 18 to 24amino acid residues that share no sequence homology with the helices ofApoA-I (Kannelis et al., 1980, J. Biol. Chem. 255(3):11464-11472;Segrest et al., 1983, J. Biol. Chem. 258:2290-2295). The sequences werespecifically designed to mimic the amphipathic helical domains of classA exchangeable apolipoproteins in terms of hydrophobic moment (Eisenberget al., 1982, Nature 299:371-374) and charge distribution (Segrest etal., 1990, Proteins 8:103-117; U.S. Pat. No. 4,643,988). One 18-residuepeptide, the “18A” peptide, was designed to be a model class-A α-helix(Segrest et al., 1990, supra). Studies with these peptides and otherpeptides having a reversed charged distribution, like the “18R” peptide,have consistently shown that charge distribution is critical foractivity; peptides with a reversed charge distribution exhibit decreasedlipid affinity relative to the 18A class-A mimics and a lower helicalcontent in the presence of lipids (Kanellis et al., 1980, J. Biol. Chem.255:11464-11472; Anantharamaiah et al., 1985, J. Biol. Chem.260:10248-10255; Chung et al., 1985, J. Biol. Chem. 260:10256-10262;Epand et al., 1987, J. Biol. Chem. 262:9389-9396; Anantharamaiah et al.,1991, Adv. Exp. Med. Biol. 285:131-140).

[0036] Other synthetic peptides sharing no sequence homology with theapolipoproteins which have been proposed with limited success includedimers and trimers of the 18A peptide (Anantharamaiah et al., 1986,Proteins of Biological Fluids 34:63-66), GALA and EALA peptides(Subbarao et al., 1988, PROTEINS: Structure, Function and Genetics3:187-198) and ID peptides (Labeur et al., 1997, Arteriosclerosis,Thrombosis and Vascular Biology 17:580-588) and the 18AM4 peptide(Brasseur et al., 1993, Biochim. Biophys. Acta 1170:1-7).

[0037] A “consensus” peptide containing 22-amino acid residues based onthe sequences of the helices of human ApoA-I has also been designed(Anantharamaiah et al., 1990, Arteriosclerosis 10(1):95-105;Venkatachalapathi et al., 1991, Mol. Conformation and Biol.Interactions, Indian Acad. Sci. B:585-596). The sequence was constructedby identifying the most prevalent residue at each position of thehypothesized helices of human ApoA-I. Like the peptides described above,the helix formed by this peptide has positively charged amino acidresidues clustered at the hydrophilic-hydrophobic interface, negativelycharged amino acid residues clustered at the center of the hydrophilicface and a hydrophobic angle of less than 180°. While a dimer of thispeptide is somewhat effective in activating LCAT, the monomer exhibitedpoor lipid binding properties (Venkatachalapathi et al., 1991, supra).

[0038] Based primarily on in vitro studies with the peptides describedabove, a set of “rules” has emerged for designing peptides which mimicthe function of apoA-I. Significantly, it is thought that an amphipathicα-helix having positively charged residues clustered at thehydrophilic-hydrophobic interface and negatively charged amino acidresidues clustered at the center of the hydrophilic face is required forlipid affinity and LCAT activation (Venkatachalapathi et al., 1991,supra). Anantharamaiah et al. have also indicated that the negativelycharged Glu residue at position 13 of the consensus 22-mer peptide,which is positioned within the hydrophobic face of the α-helix, plays animportant role in LCAT activation (Anantharamaiah et al., 1991, supra).Furthermore, Brasseur has indicated that a hydrophobic angle (pho angle)of less than 180° is required for optimal lipid-apolipoprotein complexstability, and also accounts for the formation of discoidal particleshaving the peptides around the edge of the lipid bilayer (Brasseur,1991, J. Biol. Chem. 66(24):16120-16127). Rosseneu et al. have alsoinsisted that a hydrophobic angle of less than 180° is required for LCATactivation (WO93/25581).

[0039] However, despite these “rules” to date, no one has designed orproduced a peptide as active as ApoA-I—the best having less than 40% ofthe activity of ApoA-I as measured by the LCAT activation assaydescribed herein. None of the peptide “mimetics” described in theliterature have been demonstrated to be useful as a drug.

[0040] In view of the foregoing, there is a need for the development ofa stable ApoA-I agonist that mimics the activity of ApoA-I and which isrelatively simple and cost-effective to produce. However, the “rules”for designing efficacious ApoA-I mimetics have not been unraveled andthe principles for designing organic molecules with the function ofApoA-I are unknown.

3. SUMMARY OF THE INVENTION

[0041] The invention relates to ApoA-I agonists capable of formingamphipathic α-helices that mimic the activity of ApoA-I, with specificactivities, i.e., units of activity (activation of LCAT)/unit of mass),approaching or exceeding that of the native molecule. In particular, theApoA-I agonists of the invention are peptides or peptide analogues that:form amphipathic helices (in the presence of lipids), bind lipids, formpre-β-like or HDL-like complexes, activate LCAT, increase serum levelsof HDL fractions, and promote cholesterol efflux.

[0042] The invention is based, in part, on the applicants' design anddiscovery of peptides that mimic the function of ApoA-I. The peptides ofthe invention were designed based on the supposed helical structure andamphipathic properties of the 22 amino acid consensus sequence which wasderived from the helical repeats of ApoA-I. Surprisingly, the peptidesof the invention have a specific activity well above that reported forApoA-I-derived peptides described in the literature. Indeed, someembodiments of the invention approach 100% of the activity of nativeApoA-I, whereas superagonists described herein exceed the specificactivity of ApoA-I.

[0043] One aspect of the invention is also based, in part, on theApplicants' discovery that amino acid residues 14-17 of Segrest'sconsensus 22-mers could be deleted without loss of LCAT activation. Insome cases, the deletion of amino acid residues enhances LCATactivation. In another aspect of the invention, other residues of theconsensus sequence can also be deleted with enhanced activity. Inaddition, in some embodiments, the deletion can be used in conjunctionwith alteration of amino acid residues. In some preferred embodiments,deletion of 4 residues from Segrest's consensus 22-mer peptide is usedin conjunction with changes at residue 5, 9 and 13 to a hydrophobicleucine.

[0044] The invention is illustrated by way of working examples thatdescribe the structure, preparation and use of particular amphipathicpeptides that form helices (in the presence of lipids), bind lipids,form complexes and increase LCAT activity. Based upon the structure andactivity of the exemplified embodiments, the applicants have devised aset of “rules” which can be used to design altered or mutated forms thatare also within the scope of the invention.

[0045] The invention also relates to pharmaceutical formulationscontaining such ApoA-I agonists (either as peptides or peptide-lipidcomplexes) as the active ingredient, as well as methods for preparingsuch formulations and their use to treat diseases associated withdyslipoproteinemia (e.g., cardiovascular diseases, atherosclerosis,metabolic syndrome), restenosis, or endotoxemia (e.g., septic shock).

3.1. Abbreviations

[0046] As used herein, the abbreviations for the genetically encodedL-enantiomeric amino acids are conventional and are as follows: !?One-Letter? Common? !Amino Acid? Symbol? Abbreviation Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Methionine M MetPhenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

[0047] The abbreviations used for the D-enantiomers of the geneticallyencoded amino acids are lower-case equivalents of the one-lettersymbols. For example, “R” designates L-arginine and “r” designatesD-arginine.

3.2. Definitions

[0048] As used herein, the following terms shall have the followingmeanings:

[0049] “Alkyl:” refers to a saturated branched, straight chain or cyclichydrocarbon radical. Typical alkyl groups include, but are not limitedto, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl,isopentyl, hexyl, and the like. In preferred embodiments, the alkylgroups are (C₁-C₆) alkyl.

[0050] “Alkenyl:” refers to an unsaturated branched, straight chain orcyclic hydrocarbon radical having at least one carbon-carbon doublebond. The radical may be in either the cis or trans conformation aboutthe double bond(s). Typical alkenyl groups include, but are not limitedto, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl,pentenyl, hexenyl and the like. In preferred embodiments, the alkenylgroup is (C₁-C₆) alkenyl.

[0051] “Alkynyl:” refers to an unsaturated branched, straight chain orcyclic hydrocarbon radical having at least one carbon-carbon triplebond. Typical alkynyl groups include, but are not limited to, ethynyl,propynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like. Inpreferred embodiments, the alkynyl group is (C₁-C₆) alkynyl.

[0052] “Aryl:” refers to an unsaturated cyclic hydrocarbon radicalhaving a conjugated π electron system. Typical aryl groups include, butare not limited to, penta-2,4-diene, phenyl, naphthyl, anthracyl,azulenyl, chrysenyl, coronenyl, fluoranthenyl, indacenyl, idenyl,ovalenyl, perylenyl, phenalenyl, phenanthrenyl, picenyl, pleiadenyl,pyrenyl, pyranthrenyl, rubicenyl, and the like. In preferredembodiments, the aryl group is (C₅-C₂₀) aryl, with (C₅-C₁₀) beingparticularly preferred.

[0053] “Alkaryl:” refers to a straight-chain alkyl, alkenyl or alkynylgroup wherein one of the hydrogen atoms bonded to a terminal carbon isreplaced with an aryl moiety. Typical alkaryl groups include, but arenot limited to, benzyl, benzylidene, benzylidyne, benzenobenzyl,naphthenobenzyl and the like. In preferred embodiments, the alkarylgroup is (C₆-C₂₆) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety ofthe alkaryl group is (C₁-C₆) and the aryl moiety is (C₅-C₂₀). Inparticularly preferred embodiments, the alkaryl group is (C₆-C₁₃)alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl groupis (C₁-C₃) and the aryl moiety is (C₅-C₁₀).

[0054] “Heteroaryl:” refers to an aryl moiety wherein one or more carbonatoms is replaced with another atom, such as N, P, O, S, As, Se, Si, Te,etc. Typical heteroaryl groups include, but are not limited to,acridarsine, acridine, arsanthridine, arsindole, arsindoline, carbazole,β-carboline, chromene, cinnoline, furan, imidazole, indazole, indole,indolizine, isoarsindole, isoarsinoline, isobenzofuran, isochromene,isoindole, isophosphoindole, isophosphinoline, isoquinoline,isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine,phenanthroline, phenazine, phosphoindole, phosphinoline, phthalazine,pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine,quinoxaline, selenophene, tellurophene, thiophene and xanthene. Inpreferred embodiments, the heteroaryl group is a 5-20 memberedheteroaryl, with 5-10 membered aryl being particularly preferred.

[0055] “Alkheteroaryl:” refers to a straight-chain alkyl, alkenyl oralkynyl group where one of the hydrogen atoms bonded to a terminalcarbon atom is replaced with a heteroaryl moiety. In preferredembodiments, the alkheteroaryl group is 6-26 membered alkheteroaryl,i.e., the alkyl, alkenyl or alkynyl moiety of the alkheteroaryl is(C₁-C₆) and the heteroaryl is a 5-20-membered heteroaryl. Inparticularly preferred embodiments the alkheteroaryl is 6-13 memberedalkheteroaryl, i.e., the alkyl, alkenyl or alkynyl moiety is a 5-10membered heteroaryl.

[0056] “Substituted Alkyl, Alkenyl. Alkynyl, Aryl. Alkaryl, Heteroarylor Alkheteroaryl:” refers to an alkyl, alkenyl, alkynyl, aryl, alkaryl,heteroaryl or alkheteroaryl group in which one or more hydrogen atoms isreplaced with another substituent. Preferred substituents include —OR,—SR, —NRR, —NO₂, —CN, halogen, —C(O)R, —C(O)OR and —C(O)NR, where each Ris independently hydrogen, alkyl, alkenyl, alkynyl, aryl, alkaryl,heteroaryl or alkheteroaryl.

4. BRIEF DESCRIPTION OF THE FIGURES

[0057]FIG. 1A is a Schiffer-Edmundson helical wheel diagram of anidealized amphipathic α-helix in which open circles representhydrophilic amino acid residues and shaded circles represent hydrophobicamino acid residues.

[0058]FIG. 1B is a helical net diagram of the idealized amphipathichelix of FIG. 1A.

[0059]FIG. 1C is a helical cylinder diagram of the idealized amphipathichelix of FIG. 1A.

[0060]FIG. 2A is a Schiffer-Edmundson helical wheel diagram of the corepeptide of structure (I) illustrating the amphipathicity of the helix(open circles represent hydrophilic amino acid residues, shaded circlesrepresent hydrophobic amino acid residues and partially shaded circlesrepresent either hydrophilic or hydrophobic amino acid residues).

[0061]FIG. 2B is a helical net diagram of the core peptide of structure(I) illustrating the hydrophobic face of the helix.

[0062]FIG. 2C is a helical net diagram of the core peptide of structure(I) illustrating the hydrophilic face of the helix.

[0063]FIG. 3A is a helical net diagram illustrating the hydrophilic faceof Segrest's 18A peptide (DWLKAFYDKVAEKLKEAF; SEQ ID NO:244).

[0064]FIG. 3B is a helical net diagram illustrating the hydrophilic faceof exemplary core peptide 210 (PVLDLFRELLEELKQKLK; SEQ ID NO:210).

[0065]FIG. 3C is a helical net diagram illustrating the hydrophilic faceof Segrest's consensus 22-mer peptide (PVLDEFREKLNEELEALKQKLK; SEQ IDNO:75).

[0066]FIG. 4A is a helical net diagram illustrating the hydrophobic faceof Segrest's 18A peptide (SEQ ID NO:244).

[0067]FIG. 4B is a helical net diagram illustrating the hydrophobic faceof exemplary core peptide 210 (SEQ ID NO:210).

[0068]FIG. 4C is a helical net diagram illustrating the hydrophobic faceof Segrest's consensus 22-mer peptide (SEQ ID NO:75).

[0069] FIG. SA is a Schiffer-Edmundson helical wheel diagram ofSegrest's 18A peptide (SEQ ID NO:244).

[0070]FIG. 5B is a Schiffer-Edmundson helical wheel diagram of exemplarycore peptide 210 (SEQ ID NO:210).

[0071]FIG. 6A illustrates a tertiary-order branched network of theinvention.

[0072]FIG. 6B illustrates a quaternary-order branched network of theinvention.

[0073]FIG. 6C illustrates a mixed-order branched network of theinvention.

[0074]FIG. 6D illustrates exemplary “Lys-tree” branched networks of theinvention.

[0075]FIG. 7A is a graph illustrating the differences between theobserved Hα chemical shifts and the tabulated random coil Hα chemicalshifts for peptide 210 (SEQ ID NO:210) and Segrest's consensus 22-merpeptide (SEQ ID NO:75).

[0076]FIG. 7B is a graph illustrating the differences between theobserved amide proton chemical shifts and the tabulated random coilamide proton chemical shifts for peptide 210 (SEQ ID NO:210) andSegrest's consensus 22-mer peptide (SEQ ID NO:75).

[0077]FIG. 8A is a cartoon depicting the various aggregation states andpeptide-lipid complexes that can be obtained with the ApoA-I agonists ofthe invention. Left: Multimerization process of the peptides resultingfrom the interaction of several peptide helices and leading to theformation of oligomers in conditions of defined peptide concentration,pH and ionic strength. Center: The interaction of the peptides (in anyof these states of aggregation) with lipidic entities (such as SUVs)leads to lipid reorganization. Right: By changing the lipid:peptidemolar ratio, different types of peptide-lipid complexes can be obtained,from lipid-peptide comicelles at low lipid-peptide ratios, to discoidalparticles and finally to large multilamellar complexes at increasinglyhigher lipid:peptide ratios.

[0078]FIG. 8B illustrates the generally-accepted model for discoidalpeptide-lipid complexes formed in a defined range of lipid:peptideratios. Each peptide surrounding the disc edge is in close contact withits two nearest neighbors.

5. DETAILED DESCRIPTION OF THE INVENTION

[0079] The ApoA-I agonists of the invention mimic ApoA-I function andactivity. They form amphipathic helices (in the presence of lipids),bind lipids, form pre-β-like or HDL-like complexes, activate LCAT,increase serum HDL concentration and promote cholesterol efflux. Thebiological function of the peptides correlates with their helicalstructure, or conversion to helical structures in the presence oflipids.

[0080] The ApoA-I agonists of the invention can be prepared in stablebulk or unit dosage forms, e.g., lyophilized products, that can bereconstituted before use in vivo or reformulated. The invention includesthe pharmaceutical formulations and the use of such preparations in thetreatment of hyperlipidemia, hypercholesterolemia, coronary heartdisease, atherosclerosis, and other conditions such as endotoxemiacausing septic shock.

[0081] The invention is illustrated by working examples whichdemonstrate that the ApoA-I agonists of the invention are extremelyefficient at activating LCAT, and thus promote RCT. Use of the ApoA-Iagonists of the invention in vivo in animal models results in anincrease in serum HDL concentration.

[0082] The invention is set forth in more detail in the subsectionsbelow, which describe: the composition and structure of the ApoA-Ipeptide agonists; structural and functional characterization; methods ofpreparation of bulk and unit dosage formulations; and methods of use.

5.1. Peptide Structure and Function

[0083] The ApoA-I agonists of the invention are generally peptides, oranalogues thereof, which are capable of forming amphipathic α-helices inthe presence of lipids and which mimic the activity of ApoA-I. Theagonists have as their main feature a “core” peptide composed of 15 to22 amino acid residues, preferably 18 amino acid residues, or ananalogue thereof wherein at least one amide linkage in the peptide isreplaced with a substituted amide, an isostere of an amide or an amidemimetic.

[0084] The ApoA-I agonists of the invention are based, in part, on theapplicants' surprising discovery that altering and/or deleting certainamino acid residues in the primary sequence of the 22-mer consensussequence of Venkatachalapathi et al., 1991, Mol. Conformation and Biol.Interactions, Indian Acad. Sci. B:585-596 (PVLDEFREKLNEELEALKQKLK; SEQID NO:75; hereinafter “Segrest's consensus 22-mer” or “consensus22-mer”) that were thought to be critical for activity yields syntheticpeptides which exhibit activities that approach, or in some embodimentseven exceed, the activity of native ApoA-I. In one aspect of theinvention, four amino acid residues of the consensus 22-mer, such asresidues 14-17, are deleted to form 18-mers capable of LCAT activation.In other additional embodiments, four other residues are deleted. Insome embodiments, three charged amino acid residues that were thought tobe critical for activity (Glu-5, Lys-9 and Glu-13) are replaced with ahydrophobic residue such as Leu.

[0085] While not intending to be bound by any particular theory, it isbelieved that the helix formed by the ApoA-I agonists of the inventionmore closely mimics the structural and functional properties of theamphipathic helical regions of native ApoA-I that are important foreffecting lipid-binding, cholesterol efflux and LCAT activation thandoes the α-helix formed by the ApoA-I mimetic peptides described in theliterature, thereby resulting in peptides that exhibit significantlyhigher ApoA-I-like activity than these other peptides. Indeed, whereasmany of the ApoA-I agonists of the invention approach, and in someembodiments even exceed, the activity of ApoA-I, to date, the bestpeptide ApoA-I mimics described in the literature—peptide 18AM4(EWLEAFYKKVLEKLKELF; SEQ ID NO:246) (Corinjn et al., 1993, Biochim.Biophys. Acta 1170:8-16; Labeur et al., October 1994, Arteriosclerosis:Abstract Nos. 186 and 187) and N-acetylated, C-amidated peptide 18AM4(SEQ ID NO: 239) (Brasseur, 1993, Biochim. Biophys. Acta1170:1-7)—exhibit less than 4% and 11%, respectively, of the activity ofApoA-I as measured by the LCAT activation assay described herein.

[0086] In one illustrative embodiment of the invention, the corepeptides (or analogues thereof) that compose the ApoA-I agonists of theinvention have the following structural formula (I):

X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉—X₁₀—X₁₁—X₁₂—X₁₃—X₁₄—X₁₅—X₁₆—X₁₇—X₁₈

[0087] wherein:

[0088] X₁ is Pro (P), Ala (A), Gly (G), Asn (N), Gln (Q) or D-Pro (p);

[0089] X₂ is an aliphatic amino acid;

[0090] X₃ is Leu (L);

[0091] X₄ is an acidic amino acid;

[0092] X₅ is Leu (L) or Phe (F);

[0093] X₆ is Leu (L) or Phe (F);

[0094] X₇ is a basic amino acid;

[0095] X₈ is an acidic amino acid;

[0096] X₉ is Leu (L) or Trp (W);

[0097] X₁₀ is Leu (L) or Trp (W);

[0098] X₁₁ is an acidic amino acid or Asn (N);

[0099] X₁₂ is an acidic amino acid;

[0100] X₁₃ is Leu (L), Trp (W) or Phe (F);

[0101] X₁₄ is a basic amino acid or Leu (L);

[0102] X₁₅ is Gln (Q) or Asn (N);

[0103] X₁₆ is a basic amino acid;

[0104] X₁₇ is Leu (L); and

[0105] X₁₈ is a basic amino acid.

[0106] The core peptides of structure (I) are defined, in part, in termsof amino acids of designated classes. The definitions of the variousdesignated classes are provided infra in connection with the descriptionof mutated or altered embodiments of structure (I).

[0107] In the core peptides of structure (I), the symbol “—” betweenamino acid residues X_(n) generally designates a backbone constitutivelinking function. Thus, the symbol “—” usually represents a peptide bondor amide linkage (—C(O)NH—). It is to be understood, however, that thepresent invention contemplates peptide analogues wherein one or moreamide linkages is optionally replaced with a linkage other than amide,preferably a substituted amide or an isostere of amide. Thus, while thevarious X_(n) residues within structure (I) are generally described interms of amino acids, and preferred embodiments of the invention areexemplified by way of peptides, one having skill in the art willrecognize that in embodiments having non-amide linkages, the term “aminoacid” or “residue” as used herein refers to other bifunctional moietiesbearing groups similar in structure to the side chains of the aminoacids.

[0108] Substituted amides generally include, but are not limited to,groups of the formula —C(O)NR—, where R is (C₁-C₆) alkyl, substituted(C₁-C₆) alkyl, (C₁-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, substituted (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20membered heteroaryl, substituted 5-20 membered heteroaryl or 6-26membered alkheteroaryl and substituted 6-26 membered alkheteroaryl.

[0109] Isosteres of amide generally include, but are not limited to,—CH₂NH—, —CH₂S—, —CH₂CH₂—, —CH═CH— (cis and trans), —C(O)CH₂—,—CH(OH)CH₂— and —CH₂SO—. Compounds having such non-amide linkages andmethods for preparing such compounds are well-known in the art (see,e.g., Spatola, March 1983, Vega Data Vol. 1, Issue 3; Spatola, 1983,“Peptide Backbone Modifications” In: Chemistry and Biochemistry of AminoAcids Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p.267 (general review); Morley, 1980, Trends Pharm. Sci. 1:463-468; Hudsonet al., 1979, Int. J. Prot. Res. 14:177-185 (—CH₂NH—, —CH₂CH₂—) ;Spatola et al., 1986, Life Sci. 38:1243-1249 (—CH₂—S); Hann, 1982, J.Chem. Soc. Perkin Trans. I. 1:307-314 (—CH═CH—, cis and trans); Almquistet al., 1980, J. Med. Chem. 23:1392-1398 (—COCH₂—); Jennings-White etal., Tetrahedron. Lett. 23:2533 (—COCH₂—); European Patent ApplicationEP 45665 (1982) CA 97:39405 (—CH(OH)CH₂—); Holladay et al., 1983,Tetrahedron Lett. 24:4401-4404 (—C(OH)CH₂—); and Hruby, 1982, Life Sci.31:189-199 (—CH₂—S—).

[0110] Additionally, one or more amide linkages can be replaced withpeptidomimetic or amide mimetic moieties which do not significantlyinterfere with the structure or activity of the peptides. Suitable amidemimetic moieties are described, for example, in Olson et al., 1993, J.Med. Chem. 36:3039-3049.

[0111] A critical feature of the core peptides of structure (I), istheir ability to form an amphipathic α-helix in the presence of lipids.By amphipathic is meant that the α-helix has opposing hydrophilic andhydrophobic faces oriented along its long axis, i.e., one face of thehelix projects mainly hydrophilic side chains while the opposite faceprojects mainly hydrophobic side chains. FIGS. 1A and 1B present twoillustrative views of the opposing hydrophilic and hydrophobic faces ofan exemplary idealized amphipathic α-helix. FIG. 1A is aSchiffer-Edmundson helical wheel diagram (Schiffer and Edmundson, 1967,Biophys. J. 7:121-135). In the wheel, the long axis of the helix isperpendicular to the page. Starting with the N-terminus, successiveamino acid residues (represented by circles) are radially distributedabout the perimeter of a circle at 100° intervals. Thus, amino acidresidue n+1 is positioned 100° from residue n, residue n+2 is positioned100° from residue n+1, and so forth. The 100° placement accounts for the3.6 amino acid residues per turn that are typically observed in anidealized α-helix. In FIG. 1A, the opposing hydrophilic and hydrophobicfaces of the helix are clearly visible; hydrophilic amino acids arerepresented as open circles and hydrophobic amino acid residues arerepresented as shaded circles.

[0112]FIG. 1B presents a helical net diagram of the idealizedamphipathic helix of FIG. 1A. (Lim, 1978, FEBS Lett. 89:10-14). In atypical helical net diagram, the α-helix is presented as a cylinder thathas been cut along the center of its hydrophilic face and flattened.Thus, the center of the hydrophobic face, determined by the hydrophobicmoment of the helix (Eisenberg et al., 1982, Nature 299:371-374), liesin the center of the figure and is oriented so as to rise out of theplane of the page. An illustration of the helical cylinder prior tobeing cut and flattened is depicted in FIG. 1C. By cutting the cylinderalong different planes, different views of the same amphipathic helixcan be observed, and different information about the properties of thehelix obtained.

[0113] The amphipathic nature of the α-helix formed by the core peptidesof structure (I) in the presence of lipids is illustrated in FIG. 2.FIG. 2A presents a Schiffer-Edmundson helical wheel diagram, FIG. 2Bpresents a helical net diagram illustrating the hydrophobic face andFIG. 2C presents a helical net diagram illustrating the hydrophilicface. In each of FIGS. 2A, 2B and 2C, hydrophilic residues arerepresented as open circles, hydrophobic residues as shaded circles, andresidues which can be either hydrophilic or hydrophobic as partiallyshaded circles. As will be discussed more thoroughly below inconjunction with altered or mutated forms of the peptides of structure(I), certain amino acid residues can be replaced with other amino acidresidues such that the hydrophilic and hydrophobic faces of the helixformed by the peptides may not be composed entirely of hydrophilic andhydrophobic amino acids, respectively. Thus, it is to be understood thatwhen referring to the amphipathic α-helix formed by the core peptides ofthe invention, the phrase “hydrophilic face” refers to a face of thehelix having overall net hydrophilic character. The phrase “hydrophobicface” refers to a face of the peptide having overall net hydrophobiccharacter.

[0114] While not intending to be bound by any particular theory, it isbelieved that certain structural and/or physical properties of theamphipathic helix formed by the core peptides of structure (I), areimportant for activity. These properties include the degree ofamphipathicity, overall hydrophobicity, mean hydrophobicity, hydrophobicand hydrophilic angles, hydrophobic moment, mean hydrophobic moment, andnet charge of the α-helix.

[0115] While the helical wheel diagrams of FIG. 2A provide a convenientmeans of visualizing the amphipathic nature of the core peptides ofstructure (I), the degree of amphipathicity (degree of asymmetry ofhydrophobicity) can be conveniently quantified by calculating thehydrophobic moment (μ_(H)) of the helix. Methods for calculating μ_(H)for a particular peptide sequence are well-known in the art, and aredescribed, for example in Eisenberg, 1984, Ann. Rev. Biochem.53:595-623. The actual μ_(H) obtained for a particular peptide willdepend on the total number of amino acid residues composing the peptide.Thus, it is generally not informative to directly compare μ_(H) forpeptides of different lengths.

[0116] The amphipathicities of peptides of different lengths can bedirectly compared by way of the mean hydrophobic moment (<μ_(H)>). Themean hydrophobic moment can be obtained by dividing μ_(H) by the numberof residues in the helix (i.e., <μ_(H)>=μ_(H)/N). Generally, corepeptides which exhibit a <μ> in the range of 0.55 to 0.65, as determinedusing the normalized consensus hydrophobicity scale of Eisenberg(Eisenberg, 1984, J. Mol. Biol. 179:125-142) are considered to be withinthe scope of the present invention, with a <μ_(H)> in the range of 0.58to 0.62 being preferred.

[0117] The overall or total hydrophobicity (H_(o)) of a peptide can beconveniently calculated by taking the algebraic sum of thehydrophobicities of each amino acid residue in the peptide (i.e$( {{i.e.},{H_{o} = {\sum\limits_{i = 1}^{N}H_{i}}}} ),$

[0118] where N is the number of amino acid residues in the peptide andH_(i) is the hydrophobicity of the ith amino acid residue). The meanhydrophobicity (<H_(o)>) is the hydrophobicity divided by the number ofamino acid residues (i.e., <H_(o)>=H_(o)/N). Generally, core peptidesthat exhibit a mean hydrophobicity in the range of −0.150 to −0.070, asdetermined using the normalized consensus hydrophobicity scale ofEisenberg (Eisenberg, 1984, J. Mol. Biol. 179:125-142) are considered tobe within the scope of the present invention, with a mean hydrophobicityin the range of −0.130 to −0.050 being preferred.

[0119] The total hydrophobicity of the hydrophobic face (H_(o) ^(pho))of an amphipathic helix can be obtained by taking the sum of thehydrophobicities of the hydrophobic amino acid residues which fall intothe hydrophobic angle as defined below (i.e$( {{i.e.},{H_{o}^{pho} = {\sum\limits_{i = 1}^{N}H_{i}}},} $

[0120] where H_(i) is as previously defined and N_(H) is the totalnumber of hydrophobic amino acids in the hydrophobic face). The meanhydrophobicity of the hydrophobic face (<H_(o) ^(pho)>) is H_(o)^(pho)/N_(H) where N_(H) is as defined above. Generally, core peptideswhich exhibit a <H_(o) ^(pho)>, in the range of 0.90 to 1.20, asdetermined using the consensus hydrophobicity scale of Eisenberg(Eisenberg, 1984, supra; Eisenberg et al., 1982, supra) are consideredto be within the scope of the present invention, with a <H_(o) ^(pho)>in the range of 0.950 to 1.10 being preferred.

[0121] The hydrophobic angle (pho angle) is generally defined as theangle or arc covered by the longest continuous stretch of hydrophobicamino acid residues when the peptide is arranged in theSchiffer-Edmundson helical wheel representation (i.e., the number ofcontiguous hydrophobic residues on the wheel multiplied by 20°). Thehydrophilic angle (phi angle) is the difference between 360° and the phoangle (i.e., 360°-pho angle). Those of skill in the art will recognizethat the pho and phi angles will depend, in part, on the number of aminoacid residues in the peptide. For example, referring to FIGS. 5A and 5B,it can be seen that only 18 amino acids fit around one rotation of theSchiffer-Edmundson helical wheel. Fewer amino acids leave a gap in thewheel; more amino acids cause certain positions of the wheel to beoccupied by more than one amino acid residue.

[0122] In the case of peptides containing more than 18 amino acidresidues, by “continuous” stretch of hydrophobic amino acid residues ismeant that at least one amino acid residue at positions along the wheeloccupied by two or more amino acids is a hydrophobic amino acid.

[0123] Typically, core peptides composed of 18 or fewer amino acidshaving a pho angle in the range of 120° to 160° are considered to bewithin the scope of the invention, with a pho angle in the range of 130°to 150° being preferred. Embodiments containing more than 18 amino acidstypically have a pho angle in the range of 160° to 220°, with 180° to200° being preferred.

[0124] Certain structural and/or physical characteristics of the corepeptides of structure (I) are illustrated in FIGS. 3 and 4. FIG. 3Bpresents a helical net diagram of an exemplary core peptide of theinvention, peptide 210 (PVLDLFRELLEELKQKLK; SEQ ID NO:210), illustratingthe charge distribution along the hydrophilic face of the helix. In FIG.3B, the helical cylinder has been cut along the center of thehydrophobic face and flattened. The three hydrophobic Leu (L) residuesthat replace hydrophilic residues in Segrest's consensus 22-mer(residues 5, 9 and 13) (FIG. 3C) are shaded. As can be seen in FIG. 3B,positively-charged amino acid residues are clustered at the lastC-terminal turn of the helix (the C-terminus is at the top of the page).While not intending to be bound by any particular theory, it is believedthat this cluster of basic residues at the C-terminus (residues 14, 16and 18) stabilizes the helix through charge (NH₃ ⁺)-helix dipoleelectrostatic interactions. It is also thought that stabilization occursthrough hydrophobic interactions between lysine side chains and thehelix core (see, Groebke et al., 1996, Proc. Natl. Acad. Sci. U.S.A.93:4025-4029; Esposito et al., 1997, Biopolymers 41:27-35).

[0125] With the exception of the positively-charged C-terminal cluster(residues 14, 16 and 18), negative charges are distributed on the restof the hydrophilic face, with at least one negatively charged (acidic)amino acid residue per turn, resulting in a continuous stretch ofnegative charges along the hydrophilic face of the helix.

[0126]FIG. 4B presents a helical net diagram illustrating thehydrophobic face of the amphipathic helix formed by exemplary corepeptide 210 (SEQ ID NO:210). In FIG. 4B, the helical cylinder is cutalong the center of the hydrophilic face and flattened. The hydrophobicface of the core peptide consists of two hydrophobic residues per turn,except for the last C-terminal turn, where basic residues dominate. NMRstudies indicate that amino acid residues 3, 6, 9 and 10 of an analogue22-mer peptide (PVLDLFRELLNELLEALKQKLK; SEQ ID NO:4) form a hydrophobiccluster near the N-terminus of the helix. Phe-6 is centered in thiscluster and is believed to play an important role in stabilizing thehydrophobic cluster.

[0127] While not intending to be bound by any particular theory, it isbelieved that the hydrophobic cluster formed by residues 3, 6, 9 and 10is significant in effecting lipid binding and LCAT activation.Amphipathic peptides are expected to bind phospholipids by pointingtheir hydrophobic faces towards the alkyl chains of the lipid moieties.Thus, it is believed that this highly hydrophobic cluster contributes tothe strong lipid affinities observed for the core peptides of theinvention. Since lipid binding is a prerequisite for LCAT activation, itis believed that this hydrophobic cluster is also essential for LCATactivation.

[0128] Aromatic residues are often found to be important in anchoringpeptides and proteins to lipids (De Kruijff, 1990, Biosci. Rep.10:127-130; O'Neil and De Grado, 1990, Science 250:645-651; Blondelle etal., 1993, Biochim. Biophys. Acta 1202:331-336). Thus, it is furtherbelieved that Phe-6, which is positioned at the center of thehydrophobic cluster, may also play a key role in anchoring the corepeptides of structure (I) to lipids.

[0129] Interactions between the core peptides of the invention andlipids lead to the formation of peptide-lipid complexes. As illustratedin FIG. 8A, the type of complex obtained (comicelles, discs, vesicles ormultilayers) depends on the lipid:peptide molar ratio, with comicellesgenerally being formed at low lipid:peptide molar ratios and discoidaland vesicular or multilayer complexes being formed with increasinglipid:peptide molar ratios. This characteristic has been described foramphipathic peptides (Epand, The Amphipathic Helix, 1993) and for ApoA-I(Jones, 1992, Structure and Function of Apolipoproteins, Chapter 8, pp.217-250). The lipid:peptide molar ratio also determines the size andcomposition of the complexes (see, Section 5.3.1, infra).

[0130] The long axis of the α-helix formed by the core peptides ofstructure (I) has an overall curved shape. In typical amphipathichelices, it has been found that the lengths of the hydrogen bonds of thehydrophilic and hydrophobic faces vary such that the hydrophobic side ofthe helix is concave (Barlow and Thornton, 1988, J. Mol. Biol.201:601-619; Zhou et al., 1992, J. Am. Chem. Soc. 33:11174-11183; Gesellet al., 1997, J. Biomol. NMR 9:127-135). While not intending to be boundby theory, it is believed that the overall curvature of the hydrophobicface of the helix may be important in binding discoidal complexes—acurved helix permits the peptide to “fit” better around the edges ofdiscoidal particles, thereby increasing the stability of thepeptide-disc complex.

[0131] In the generally accepted structural model of ApoA-I, theamphipathic α-helices are packed around the edge of the discoidal HDL(see, FIG. 8B). In this model, the helices are assumed to be alignedwith their hydrophobic faces pointing towards the lipid acyl chains(Brasseur et al., 1990, Biochim. Biophys. Acta 1043:245-252). Thehelices are arranged in an antiparallel fashion, and a cooperativeeffect between the helices is thought to contribute to the stability ofthe discoidal HDL complex (Brasseur et al., supra). It has been proposedthat one factor that contributes to the stability of the HDL discoidalcomplex is the existence of ionic interactions between acidic and basicresidues resulting in the formation of intermolecular salt bridges orhydrogen bonds between residues on adjacent anti-parallel helices. Inthis model, the peptides are considered not as a single entity, but asin interaction with at least two other neighboring peptide molecules(FIG. 8B).

[0132] It is also generally accepted that intramolecular hydrogen bondor salt bridge formation between acidic and basic residues,respectively, at positions i and i+3 of the helix stabilize the helicalstructure (Marqusee et al., 1985, Proc. Natl. Acad. Sci. USA84(24):8898-8902). One positive charge is located at residue 7potentially contributing to helix stability by forming a salt bridgewith an acidic residue one turn away.

[0133] Thus, additional key features of the core peptides of structure(I) are their ability to form intermolecular hydrogen-bonds with oneanother when aligned in an antiparallel fashion with their hydrophobicfaces pointing in the same direction, such as would be the case when thepeptides are bound to lipids and also their ability to formintermolecular hydrogen bonds or salt bridges near the N- and C-terminiof the helix. It is believed that the ability of the core peptides ofstructure (I) to closely pack and ionically interact to form intra-and/or inter-molecular salt bridges and/or hydrogen bonds when bound tolipids in an antiparallel fashion is an important feature of the corepeptides of the invention.

[0134] The ability of the core peptides to form favorable intermolecularpeptide-peptide interactions is also thought to be of relevance in theabsence of lipids. The core peptides of the invention self-associate,due in part to their high <μ_(H)>, <H_(o)> and hydrophobic angle (see,TABLE I, infra). The self-association phenomenon depends on theconditions of pH, peptide concentration and ionic strength, and canresult in several states of association, from monomeric to severalmultimeric forms (FIG. 10A). The hydrophobic core of peptide aggregatesfavors hydrophobic interactions with lipids. The ability of the peptidesto aggregate even at very low concentrations may favor their binding tolipids. It is thought that in the core of the peptide aggregatespeptide-peptide interactions also occur and may compete withlipid-peptide interactions.

[0135] In addition to the above-described properties, other parametersare thought to be important for activity as well, including the totalnumber of hydrophobic residues, the total number of charged residues,and the net charge of the peptides.

[0136] A summary of the preferred physical and structural properties ofthe core peptides of structure (I) is provided in TABLE I, below: TABLEI PHYSICAL PROPERTIES OF PREFERRED ApoA-I AGONISTS OF STRUCTURE (I)PROPERTY RANGE PREFERRED RANGE % hydrophobic amino 40-70 50-60 acids<H_(o)> −0.150 to −0.070 −0.130 to −0.050 <H_(o) ^(pho)> 0.90-1.2 0.95-1.10 <μ_(H)> 0.55-0.65 0.58-0.62 pho angle 120°-160° 130°-150° #positively 3-5 4 charged amino acids # negatively 3-5 4 charged aminoacids net charge −1 to +1 0 hydrophobic cluster positions 3, 6, 9, 10are hydrophobic amino acids acidic cluster at least 1 acidic amino acidper turn except for last 5 C-terminal amino acids basic cluster at least2 basic amino acids in last 5 C-terminal amino acids

[0137] The properties of the amphipathic α-helices formed by the corepeptides of the invention differ significantly from the properties ofclass A amphipathic α-helices, particularly the class A α-helix ofSegrest's 18A and consensus 22-mer peptides. These differences areillustrated with exemplary core peptide 210 (SEQ ID NO:210) in FIGS.3-5.

[0138] Referring to FIGS. 4A-4C, it can be seen that the hydrophobicface of peptide 210 has much greater hydrophobic character than thehydrophobic face of Segrest's 18A peptide or consensus 22-mer. Inparticular, residues 9 and 13 (shaded region of FIG. 4B) are hydrophobicLeu (L) residues in peptide 210 (SEQ ID NO:210) as compared to chargedresidues in the 18A peptide (SEQ ID NO:244) and the consensus 22-mer(SEQ ID NO:75). The replacement of these two charged residues inSegrest's 18A and consensus 22-mer peptides with hydrophobic Leu (L)residues leads to significant differences in the amphipathicity,hydrophobicity, pho angle and other properties of the helix.

[0139] A comparison of the physical and structural properties of peptide210 (SEQ ID NO:210) and Segrest's 18A peptide (SEQ ID NO:244) andconsensus 22-mer peptide (SEQ ID NO:75) is provided in TABLE II, below:TABLE II COMPARISON OF PROPERTIES OF EXEMPLARY CORE PEPTIDE 210 (SEQ IDNO:210) WITH SEGREST'S CONSENSUS 22-MER (SEQ ID NO:75) AND 18A PEPTIDE(SEQ ID NO:244) CONSENSUS PEPTIDE PROPERTY 18A 22-MER 210 # amino acids18 22 18 # hydrophilic 9 13 9 amino acids # hydrophobic 9 9 9 aminoacids % hydrophobic 50 41 50 amino acids <H_(o)> −0.43 −0.293 −0.125<H_(o) ^(pho)> 0.778 0.960 1.081 <μ_(H)> 0.485 0.425 0.597 pho angle100° 100° 140° # positively 4 5 4 charged amino acids # negatively 4 6 4charged amino acids net charge 0 −1 0

[0140] These differences in properties lead to significant differencesin activity. Whereas Segrest's 18A peptide (SEQ ID NO:244) and consensus22-mer peptide (SEQ ID NO:75) exhibit only 5% and 10% LCAT activation,respectively, as compared with native ApoA-I in the assays describedherein, peptide 210 (SEQ ID NO:210) exhibits 46% activation as comparedwith native ApoA-I in the same assays. Peptide 193 (SEQ ID NO:193),which is the N-terminal acetylated and C-terminal amidated form ofpeptide 210, exhibits 96% LCAT activation in the same assay.

[0141] Certain amino acid residues in the core peptides of structure (I)can be replaced with other amino acid residues without significantlydeleteriously affecting, and in many cases even enhancing, the activityof the peptides. Thus, also contemplated by the present invention arealtered or mutated forms of the core peptides of structure (I) whereinat least one defined amino acid residue in the structure is substitutedwith another amino acid residue. As one of the critical featuresaffecting the activity of the core peptides of the invention is believedto be their ability to form α-helices in the presence of lipids thatexhibit the amphipathic and other properties described above, it will berecognized that in preferred embodiments of the invention, the aminoacid substitutions are conservative, i.e., the replacing amino acidresidue has physical and chemical properties that are similar to theamino acid residue being replaced.

[0142] For purposes of determining conservative amino acidsubstitutions, the amino acids can be conveniently classified into twomain categories—hydrophilic and hydrophobic—depending primarily on thephysical-chemical characteristics of the amino acid side chain. Thesetwo main categories can be further classified into subcategories thatmore distinctly define the characteristics of the amino acid sidechains. For example, the class of hydrophilic amino acids can be furthersubdivided into acidic, basic and polar amino acids. The class ofhydrophobic amino acids can be further subdivided into apolar andaromatic amino acids. The definitions of the various categories of aminoacids that define structure (I) are as follows:

[0143] “Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Thr(T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) andArg (R).

[0144] “Acidic Amino Acid” refers to a hydrophilic amino acid having aside chain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

[0145] “Basic Amino Acid” refers to a hydrophilic amino acid having aside chain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg (R) and Lys (K).

[0146] “Polar Amino Acid” refers to a hydrophilic amino acid having aside chain that is uncharged at physiological pH, but which has at leastone bond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q) Ser (S) and Thr (T).

[0147] “Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophobic amino acids include Pro(P), Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly(G) and Tyr (Y).

[0148] “Aromatic Amino Acid” refers to a hydrophobic amino acid with aside chain having at least one aromatic or heteroaromatic ring. Thearomatic or heteroaromatic ring may contain one or more substituentssuch as —OH, —SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂, —NHR, —NRR,—C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR and the likewhere each R is independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,(C₁-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl,substituted (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₅-C₂₀) aryl,(C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 memberedheteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).

[0149] “Nonpolar Amino Acid” refers to a hydrophobic amino acid having aside chain that is uncharged at physiological pH and which has bonds inwhich the pair of electrons shared in common by two atoms is generallyheld equally by each of the two atoms (i.e., the side chain is notpolar). Genetically encoded apolar amino acids include Leu (L), Val (V),Ile (I), Met (M), Gly (G) and Ala (A).

[0150] “Aliphatic Amino Acid” refers to a hydrophobic amino acid havingan aliphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala (A), Val (V), Leu (L) and Ile (I).

[0151] The amino acid residue Cys (C) is unusual in that it can formdisulfide bridges with other Cys (C) residues or othersulfanyl-containing amino acids. The ability of Cys (C) residues (andother amino acids with -SH containing side chains) to exist in a peptidein either the reduced free -SH or oxidized disulfide-bridged formaffects whether Cys (C) residues contribute net hydrophobic orhydrophilic character to a peptide. While Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg, 1984, supra), it is to be understood that forpurposes of the present invention Cys (C) is categorized as a polarhydrophilic amino acid, notwithstanding the general classificationsdefined above.

[0152] As will be appreciated by those of skill in the art, theabove-defined categories are not mutually exclusive. Thus, amino acidshaving side chains exhibiting two or more physical-chemical propertiescan be included in multiple categories. For example, amino acid sidechains having aromatic moieties that are further substituted with polarsubstituents, such as Tyr (Y), may exhibit both aromatic hydrophobicproperties and polar or hydrophilic properties, and can therefore beincluded in both the aromatic and polar categories. The appropriatecategorization of any amino acid will be apparent to those of skill inthe art, especially in light of the detailed disclosure provided herein.

[0153] Certain amino acid residues, called “helix breaking” amino acids,have a propensity to disrupt the structure of α-helices when containedat internal positions within the helix. Amino acid residues exhibitingsuch helix-breaking properties are well-known in the art (see, e.g.,Chou and Fasman, Ann. Rev. Biochem. 47:251-276) and include Pro (P), Gly(G) and potentially all D-amino acids (when contained in an L-peptide;conversely, L-amino acids disrupt helical structure when contained in aD-peptide). While these helix-breaking amino acid residues fall into thecategories defined above, these residues should not be used tosubstitute amino acid residues at internal positions within thehelix—they should only be used to substitute 1-3 amino acid residues atthe N-terminus and/or C-terminus of the peptide.

[0154] While the above-defined categories have been exemplified in termsof the genetically encoded amino acids, the amino acid substitutionsneed not be, and in certain embodiments preferably are not, restrictedto the genetically encoded amino acids. Indeed, many of the preferredpeptides of structure (I) contain genetically non-encoded amino acids.Thus, in addition to the naturally occurring genetically encoded aminoacids, amino acid residues in the core peptides of structure (I) may besubstituted with naturally occurring non-encoded amino acids andsynthetic amino acids.

[0155] Certain commonly encountered amino acids which provide usefulsubstitutions for the core peptides of structure (I) include, but arenot limited to, β-alanine (β-Ala) and other omega-amino acids such as3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyricacid and so forth; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid(Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly);ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA);t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg);cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal);4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F));3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F));penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MSO);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH₂));N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hphe)and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro),N-methylated amino acids and peptoids (N-substituted glycines).

[0156] The classifications of the genetically encoded and commonnon-encoded amino acids according to the categories defined above aresummarized in TABLE III, below. It is to be understood that TABLE III isfor illustrative purposes only and does not purport to be an exhaustivelist of amino acid residues that can be used to substitute the corepeptides described herein. Other amino acid residues not specificallymentioned herein can be readily categorized based on their observedphysical and chemical properties in light of the definitions providedherein. TABLE III CLASSIFICATIONS OF COMMONLY ENCOUNTERED AMINO ACIDSGenetically Non-Genetically Classification Encoded Encoded HydrophobicAromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4- Cl), Phe(2-F), Phe(3-F),Phe(4-F), hPhe Apolar L, V, I, M, G, A, P t-BuA, t-BuG, MeIle, Nle,MeVal, Cha, McGly, Aib Aliphatic A, V, L, I b-Ala, Dpr, Aib, Aha, MeGly,t-BuA, t-BuG, Metle, Cha, Nle, MeVal Hydrophilic Acidic D, E Basic H, K,R Dpr, Orn, hArg, Phe(p-NH2), Dbu, Dab Polar C, Q, N, S, T Cit, AcLys,MSO, bAla, hSer Helix-Breaking P, G D-Pro and other D-amino acids (inL-peptides)

[0157] While in most instances, the amino acids of the core peptides ofstructure (I) will be substituted with L-enantiomeric amino acids, thesubstitutions are not limited to L-enantiomeric amino acids. Thus, alsoincluded in the definition of “mutated” or “altered” forms are thosesituations where at least one L-amino acid is replaced with an identicalD-amino acid (e.g., L-Arg→D-Arg) or with a D-amino acid of the samecategory or subcategory (e.g., L-Arg→D-Lys), and vice versa. Indeed, incertain preferred embodiments that are suitable for oral administrationto animal subjects, the peptides may advantageously be composed of atleast one D-enantiomeric amino acid. Peptides containing such D-aminoacids are thought to be more stable to degradation in the oral cavity,gut or serum than are peptides composed exclusively of L-amino acids.

[0158] As noted above, D-amino acids tend to disrupt the structure ofα-helices when contained at internal positions of an α-helicalL-peptide. As a consequence, D-amino acids should not be used tosubstitute internal L-amino acids; D-amino acid substitutions should belimited to 1-3 amino acid residues at the N-terminus and/or C-terminusof the peptide. Preferably, only the amino acid at the N- and/orC-terminus is substituted with a D-amino acid.

[0159] Using the amino acid residue classifications described above inconjunction with the Schiffer-Edmundson helical wheel and helical netdiagram presentations of the core peptides of structure (I), as well asthe detailed description of the desired properties provided herein,altered or mutated forms of the core peptides of structure (I) thatsubstantially retain the amphipathic and other properties of the helix,and which are therefore considered to be within the scope of the presentinvention, can be readily obtained.

[0160] In a preferred embodiment of the invention, altered or mutatedforms of the core peptides of structure (I) are obtained by fixing thehydrophilic or hydrophobic residues according to structure (I) andsubstituting at least one non-fixed residue with another amino acid,preferably conservatively, i.e., with another amino acid of the samecategory or sub-category. The residues composing the basic and/orhydrophobic clusters can also be fixed according to structure (I), andat least one non-fixed residue substituted, preferably conservatively.

[0161] In another preferred embodiment, altered or mutated forms of thecore peptides of structure (I) are obtained by fixing the hydrophilicamino acid residues positioned within the hydrophilic face of the helixaccording to structure (I) and substituting at least one non-fixed aminoacid residue with another amino acid, preferably with another amino acidresidue of the same category or sub-category. Referring to FIG. 2A, itcan be seen that residues 1, 4, 7, 8, 11, 12, 14, 15 and 18 arepositioned within the hydrophilic face of the amphipathic helix formedby the core peptides of structure (I). Of these residues, all arehydrophilic except for residue 1, which may be either hydrophilic orhydrophobic. Thus, in one preferred embodiment, residues 4, 7, 8, 11,12, 14, 15 and 18 are fixed according to structure (I) and at least oneof residues 1, 2, 3, 5, 6, 9, 10, 13, 16 and 17 is substituted withanother amino acid of the same category, preferably with another aminoacid of the same sub-category. Alternatively, residue 1 is also fixedaccording to structure (I) and at least one of residues 2, 3, 5, 6, 9,10, 13, 16 and 17 is substituted.

[0162] In a particularly preferred embodiment, the C-terminal basiccluster (residues 14, 16 and 18) is also fixed according to structure(I), and only residues 2, 3, 5, 6, 9, 10, 13 and/or 17 are substituted.

[0163] In another particularly preferred embodiment, the hydrophobiccluster (residues 3, 6, 9 and 10) is also fixed according to structure(I), and only residues 2, 5, 13, 16 and/or 17 are substituted.

[0164] In still another particularly preferred embodiment, both thebasic and hydrophobic clusters are fixed and only residues 2, 5, 13and/or 17 are substituted.

[0165] In another preferred embodiment of the invention, altered ormutated forms of the core peptides of the invention are obtained byfixing the hydrophobic amino acid residues positioned within thehydrophobic face of the helix and substituting at least one non-fixedamino acid residue with another amino acid residue, preferably withanother residue of the same category or sub-category.

[0166] Referring to FIG. 2A, it can be seen that residues 2, 3, 5, 6, 9,10, 13, 16 and 17 are positioned within the hydrophobic face. Of these,all are hydrophobic except for residue 16, which is hydrophilic. Thus,in one preferred embodiment residues 2, 3, 5, 6, 9, 10, 13 and 17 arefixed according to structure (I) and at least one of residues 1, 4, 7,8, 11, 12, 14, 15, 16 and 18 is substituted with another amino acidresidue, preferably with another amino acid of the same category orsubcategory.

[0167] In a particularly preferred embodiment, the C-terminal basiccluster (residues 14, 16 and 18) is also fixed, and only residues 1, 4,7, 8, 11, 12 and/or 15 are substituted.

[0168] In another embodiment, altered or mutated forms of the peptidesof structure (I) are obtained by fixing all of the amino acid residuesresiding within the hydrophobic or hydrophilic face of the helix andsubstituting, preferably conservatively, at least one amino acid residueresiding in the other face with another amino acid residue. The residuescomprising the hydrophobic cluster and/or the basic cluster may also beoptionally fixed according to structure (I), as previously discussed.

[0169] In another embodiment of the invention, the altered or mutatedforms of structure (I) are obtained by substituting at least one aminoacid with a non-conservative amino acid. Those of skill in the art willrecognize that such substitutions should not substantially alter theamphipathic and/or structural properties of the helix discussed, supra.Thus, in certain instances it may be desirable to substitute one or morepairs of amino acids so as to preserve the net properties of the helix.Further guidance for selecting appropriate amino acid substitutions isprovided by the peptide sequences listed in TABLE X (see, Section 8.3,infra).

[0170] In still another embodiment of the invention, the first one tofour amino acid residues at the N-terminus and/or C-terminus of the corepeptides of structure (I) are substituted with one or more amino acidresidues, or one or more peptide segments, that are known to conferstability to regions of α-helical secondary structure (“end-cap”residues or segments). Such end-cap residues and segments are well-knownin the art (see, e.g., Richardson and Richardson, 1988, Science240:1648-1652; Harper et al., 1993, Biochemistry 32(30) :7605-7609;Dasgupta and Bell, 1993, Int. J. Peptide Protein Res. 41:499-511; Sealeet al., 1994, Protein Science 3:1741-1745; Doig et al., 1994,Biochemistry 33:3396-3403; Zhou et al., 1994, Proteins 18:1-7; Doig andBaldwin, 1995, Protein Science 4:1325-1336; Odaert et al., 1995,Biochemistry 34:12820-12829; Petrukhov et al., 1996, Biochemistry35:387-397; Doig et al., 1997, Protein Science 6:147-155).Alternatively, the first one to four N-terminal and/or C-terminal aminoacid residues of structure (I) can be replaced with peptidomimeticmoieties that mimic the structure and/or properties of end-cap residuesor segments. Suitable end-cap mimetics are well-known in the art, andare described, for example, in Richardson and Richardson, 1988, Science240:1648-1652; Harper et al., 1993, Biochemistry 32(30):7605-7609;Dasgupta and Bell, 1993, Int. J. Peptide Protein Res. 41:499-511; Sealeet al., 1994, Protein Science 3:1741-1745; Doig et al., 1994,Biochemistry 33:3396-3403; Zhou et al., 1994, Proteins 18:1-7; Doig andBaldwin, 1995, Protein Science 4:1325-1336; Odaert et al., 1995,Biochemistry 34:12820-12829; Petrukhov et al., 1996, Biochemistry35:387-397; Doig et al., 1997, Protein Science 6:147-155).

[0171] While structure (I) contains 18 specified amino acid residuepositions, it is to be understood that the core peptides of theinvention can contain fewer than 18 amino acid residues. Indeed,truncated or internally deleted forms of structure (I) containing as fewas 14-15 amino acid residues that substantially retain the overallcharacteristics and properties of the amphipathic helix formed by thecore peptides of structure (I) are considered to be within the scope ofthe present invention.

[0172] Truncated forms of the peptides of structure (I) are obtained bydeleting one or more amino acids from the N- and/or C-terminus ofstructure (I). Internally deleted forms of structure (I) are obtained bydeleting one or more amino acids from internal positions within thepeptide of structure (I). The internal amino acid residues deleted mayor may not be consecutive residues.

[0173] Those of skill in the art will recognize that deleting aninternal amino acid residue from a core peptide of structure (I) willcause the plane of the hydrophilic-hydrophobic interface of the helix torotate by 100° at the point of the deletion. As such rotations cansignificantly alter the amphipathic properties of the resultant helix,in a preferred embodiment of the invention amino acid residues aredeleted so as to substantially retain the alignment of the plane of thehydrophilic-hydrophobic interface along the entire long axis of thehelix.

[0174] This can be conveniently achieved by deleting a sufficient numberof consecutive or non-consecutive amino acid residues such that onecomplete helical turn is deleted. An idealized α-helix contains 3.6residues per turn. Thus, in a preferred embodiment, groups of 3-4consecutive or non-consecutive amino acid residues are deleted. Whether3 amino acids or 4 amino acids are deleted will depend upon the positionwithin the helix of the first residue to be deleted. Determining theappropriate number of consecutive or non-consecutive amino acid residuesthat constitute one complete helical turn from any particular startingpoint within an amphipathic helix is well within the capabilities ofthose of skill in the art.

[0175] Due to the surmised importance of the basic cluster at theC-terminus of the core peptides of structure (I) in stabilizing thehelix and the importance of the hydrophobic cluster in effecting lipidbinding and LCAT activation, in preferred embodiments of the invention,residues comprising the basic and hydrophobic clusters are not deleted.Thus, in preferred embodiments, residues 14, 16 and 18 (basic cluster)and residues 3, 6, 9 and 10 (hydrophobic cluster) are not deleted.

[0176] The core peptides of structure (I) can also be extended at one orboth termini or internally with additional amino acid residues that donot substantially interfere with, and in some embodiments even enhance,the structural and/or functional properties of the peptides. Indeed,extended core peptides containing as many as 19, 20, 21, 22 or even moreamino acid residues are considered to be within the scope of the presentinvention. Preferably, such extended peptides will substantially retainthe net amphipathicity and other properties of the peptides of structure(I). Of course, it will be recognized that adding amino acids internallywill rotate the plane of the hydrophobic-hydrophilic interface at thepoint of the insertion in a manner similar to that described above forinternal deletions. Thus, the considerations discussed above inconnection with internal deletions apply to internal additions, as well.

[0177] In one embodiment, the core peptides are extended at the N-and/or C-terminus by least one helical turn. Preferably, such extensionswill stabilize the helical secondary structure in the presence oflipids, such as the end-cap amino acids and segments previouslydescribed.

[0178] In a particularly preferred embodiment, the core peptide ofstructure (I) is extended at the C-terminus by a single basic amino acidresidue, preferably Lys (K).

[0179] Also included within the scope of the present invention are“blocked” forms of the ApoA-I agonist, i.e., forms of the ApoA-Iagonists in which the N- and/or C-terminus is blocked with a moietycapable of reacting with the N-terminal —NH₂ or C-terminal —C(O)OH. Ithas been discovered that removing the N- and/or C-terminal charges ofthe ApoA-I agonists of the invention containing 18 or fewer amino acidresidues (by synthesizing N-acylated peptideamides/ester/hydrazides/alcohols and substitutions thereof) results inagonists which approach, and in some embodiments even exceed, theactivity of the unblocked form of the agonist. For example, whilepeptide 210 (SEQ ID NO:210) exhibits 46% LCAT activation as comparedwith native ApoA-I, an N- and C-terminal blocked form of this peptide,peptide 193 (SEQ ID NO:193), exhibits 96% LCAT activation in the sameassay. In some embodiments containing 22 amino acids, blocking the N- orC-terminus results in ApoA-I agonists which exhibit lower activity thanthe unblocked forms. However, blocking both the N- and C-termini ofApoA-I agonists composed of 22 amino acids is expected to restoreactivity. Thus, in a preferred embodiment of the invention, either theN- and/or C-terminus (preferably both termini) of core peptidescontaining 18 or fewer amino acids are blocked, whereas the N- andC-termini of peptides containing more than 18 amino acids are eitherboth blocked or both unblocked. Typical N-terminal blocking groupsinclude RC(O)—, where R is —H, (C₁-C₆) alkyl, (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, (C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl or6-26 membered alkheteroaryl. Preferred N-terminal blocking groupsinclude acetyl, formyl and dansyl. Typical C-terminal blocking groupsinclude —C(O)NRR and —C(O)OR, where each R is independently defined asabove. Preferred C-terminal blocking groups include those where each Ris independently methyl. While not intending to be bound by anyparticular theory, it is believed that such terminal blocking groupsstabilize the α-helix in the presence of lipids (see, e.g.,Venkatachelapathi et al., 1993, PROTEINS: Structure, Function andGenetics 15:349-359).

[0180] The native structure of ApoA-I contains eight helical units thatare thought to act in concert to bind lipids (Nakagawa et al., 1985, J.Am. Chem. Soc. 107:7087-7092; Anantharamaiah et al., 1985, J. Biol.Chem. 260:10248-10262; Vanloo et al., 1991, J. Lipid Res. 32:1253-1264;Mendez et al., 1994, J. Clin. Invest. 94:1698-1705; Palgunari et al.,1996, Arterioscler. Thromb. Vasc. Biol. 16:328-338; Demoor et al., 1996,Eur. J. Biochem. 239:74-84). Thus, also included in the presentinvention are ApoA-I agonists comprised of dimers, trimers, tetramersand even higher order polymers (“multimers”) of the core peptidesdescribed herein. Such multimers may be in the form of tandem repeats,branched networks or combinations thereof. The core peptides may bedirectly attached to one another or separated by one or more linkers.

[0181] The core peptides that comprise the multimers may be the peptidesof structure (I), analogues of structure (I), mutated forms of structure(I), truncated or internally deleted forms of structure (I), extendedforms of structure (I) and/or combinations thereof. The core peptidescan be connected in a head-to-tail fashion (i.e., N-terminus toC-terminus), a head-to-head fashion, (i.e., N-terminus to N-terminus), atail-to-tail fashion (i.e., C-terminus to C-terminus), or combinationsthereof.

[0182] In one embodiment of the invention, the multimers are tandemrepeats of two, three, four and up to about ten core peptides.Preferably, the multimers are tandem repeats of from 2 to 8 corepeptides. Thus, in one embodiment, the ApoA-I agonists of the inventioncomprise multimers having the following structural formula:

HHLL_(m)—HH_(n)LL_(m)—HH  (II)

[0183] wherein:

[0184] each m is independently an integer from 0 to 1, preferably 1;

[0185] n is an integer from 0 to 10, preferably 0 to 8;

[0186] each “HH” independently represents a core peptide or peptideanalogue of structure (I) or a mutated, truncated, internally deleted orextended form thereof as described herein;

[0187] each “LL” independently represents a linker; and

[0188] each “—” independently designates a covalent linkage.

[0189] In structure (II), the linker LL can be any bifunctional moleculecapable of covalently linking two peptides to one another. Thus,suitable linkers are bifunctional molecules in which the functionalgroups are capable of being covalently attached to the N- and/orC-terminus of a peptide. Functional groups suitable for attachment tothe N- or C-terminus of peptides are well known in the art, as aresuitable chemistries for effecting such covalent bond formation.

[0190] The linker may be flexible, rigid or semi-rigid, depending on thedesired properties of the multimer. Suitable linkers include, forexample, amino acid residues such as Pro or Gly or peptide segmentscontaining from about 2 to about 5, 10, 15 or 20 or even more aminoacids, bifunctional organic compounds such as H₂N(CH₂)_(n)COOH where nis an integer from 1 to 12, and the like. Examples of such linkers, aswell as methods of making such linkers and peptides incorporating suchlinkers are well-known in the art (see, e.g., Hunig et al., 1974, Chem.Ber. 100:3039-3044; Basak et al., 1994, Bioconjug. Chem. 5(4):301-305).

[0191] In a preferred embodiment of the invention, the tandem repeatsare internally punctuated by a single proline residue. To this end, inthose instances where the core peptides are terminated at their N- orC-terminus with proline, such as, e.g., where X₁ in structure (I) is Pro(P) or D-Pro (p), m in structure (II) is preferably 0. In thoseinstances where the core peptides do not contain an N- or C-terminalproline, LL is preferably Pro (P) or D-Pro (p) and m is preferably 1.

[0192] In certain embodiments of the invention, it may be desirable toemploy cleavable linkers that permit the release of one or more helicalsegments (HH) under certain conditions. Suitable cleavable linkersinclude peptides having amino acid sequences that are recognized byproteases, oligonucleotides that are cleaved by endonucleases andorganic compounds that can be cleaved via chemical means, such as underacidic, basic or other conditions. Preferably, the cleavage conditionswill be relatively mild so as not to denature or otherwise degrade thehelical segments and/or non-cleaved linkers composing the multimericApoA-I agonists.

[0193] Peptide and oligonucleotide linkers that can be selectivelycleaved, as well as means for cleaving the linkers are well known andwill be readily apparent to those of skill in the art. Suitable organiccompound linkers that can be selectively cleaved will be apparent tothose of skill in the art, and include those described, for example, inWO 94/08051, as well as the references cited therein.

[0194] In a preferred embodiment, the linkers employed are peptides thatare substrates for endogenous circulatory enzymes, thereby permittingthe multimeric ApoA-I agonists to be selectively cleaved in vivo.Endogenous enzymes suitable for cleaving the linkers include, forexample, proapolipoprotein A-I propeptidase. Appropriate enzymes, aswell as peptide segments that act as substrates for such enzymes, arewell-known in the art (see, e.g., Edelstein et al., 1983, J. Biol. Chem.258:11430-11433; Zanis, 1983, Proc. Natl. Acad. Sci. USA 80:2574-2578).

[0195] As discussed above, a key feature of the core peptides of theinvention is their ability to form intermolecular hydrogen-bonds or saltbridges when arranged in an antiparallel fashion. Thus, in a preferredembodiment of the invention, linkers of sufficient length andflexibility are used so as to permit the helical segments (HH) ofstructure (II) to align in an antiparallel fashion and formintermolecular hydrogen-bonds or salt bridges in the presence of lipids.

[0196] Linkers of sufficient length and flexibility include, but are notlimited to, Pro (P), Gly (G), Cys-Cys, H₂N—(CH₂)_(n)—C(O)OH where n is 1to 12, preferably 4 to 6; H₂N-aryl-C(O)OH and carbohydrates.

[0197] Alternatively, as the native apolipoproteins permit cooperativebinding between antiparallel helical segments, peptide linkers whichcorrespond in primary sequence to the peptide segments connectingadjacent helices of the native apolipoproteins, including, for example,ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE and ApoJcan be conveniently used to link the core peptides. These sequences arewell known in the art (see, e.g., Rosseneu et al., “Analysis of thePrimary and of the Secondary Structure of the Apolipoproteins,” In:Structure and Function of Lipoproteins, Ch. 6, 159-183, CRC Press, Inc.,1992).

[0198] Other linkers which permit the formation of intermolecularhydrogen bonds or salt bridges between tandem repeats of antiparallelhelical segments include peptide reverse turns such as β-turns andγ-turns, as well as organic molecules that mimic the structures ofpeptide β-turns and/or γ-turns. Generally, reverse turns are segments ofpeptide that reverse the direction of the polypeptide chain so as toallow a single polypeptide chain to adopt regions of antiparallelβ-sheet or antiparallel α-helical structure. β-turns generally arecomposed of four amino acid residues and γ-turns are generally composedof three amino acid residues.

[0199] The conformations and sequences of many peptide β-turns have beenwell-described in the art and include, by way of example and notlimitation, type-I, type-I′, type-II, type-II′, type-III, type-III′,type-IV, type-V, type-V′, type-VIa, type-VIb, type-VII and type-VIII(see, Richardson, 1981, Adv. Protein Chem. 34:167-339; Rose et al.,1985, Adv. Protein Chem. 37:1-109; Wilmot et al., 1988, J. Mol. Biol.203:221-232; Sibanda et al., 1989, J. Mol. Biol. 206:759-777; Tramontanoet al., 1989, Proteins: Struct. Funct. Genet. 6:382-394).

[0200] The specific conformations of short peptide turns such as β-turnsdepend primarily on the positions of certain amino acid residues in theturn (usually Gly, Asn or Pro). Generally, the type-I β-turn iscompatible with any amino acid residue at positions 1 through 4 of theturn, except that Pro cannot occur at position 3. Gly predominates atposition 4 and Pro predominates at position 2 of both type-I and type-IIturns. Asp, Asn, Ser and Cys residues frequently occur at position 1,where their side chains often hydrogen-bond to the NH of residue 3.

[0201] In type-II turns, Gly and Asn occur most frequently at position3, as they adopt the required backbone angles most easily. Ideally,type-I′ turns have Gly at positions 2 and 3, and type-II′ turns have Glyat position 2. Type-III turns generally can have most amino acidresidues, but type-III′ turns usually require Gly at positions 2 and 3.Type-VIa and VIb turns generally have a cis peptide bond and Pro as aninternal residue. For a review of the different types and sequences ofβ-turns in proteins and peptides the reader is referred to Wilmot etal., 1988, J. Mol. Biol. 203:221-232.

[0202] The conformation and sequences of many peptide γ-turns have alsobeen well-described in the art (see, e.g., Rose et al., 1985, Adv.Protein Chem. 37:1-109; Wilmer-White et al., 1987, Trends Biochem. Sci.12:189-192; Wilmot et al., 1988, J. Mol. Biol. 203:221-232; Sibanda etal., 1989, J. Mol. Biol. 206:759-777; Tramontano et al., 1989, Proteins:Struct. Funct. Genet. 6:382-394). All of these types of β-turns andγ-turn structures and their corresponding sequences, as well as laterdiscovered peptide β-turns and γ-turn structures and sequences, arespecifically contemplated by the invention.

[0203] Alternatively, the linker (LL) may comprise an organic moleculeor moiety that mimics the structure of a peptide β-turn or γ-turn. Suchβ-turn and/or γ-turn mimetic moieties, as well as methods forsynthesizing peptides containing such moieties, are well known in theart, and include, among others, those described in Giannis and Kolter,1993 Angew. Chem. Intl. Ed. Eng. 32:1244-1267; Kahn et al., 1988, J.Molecular Recognition 1:75-79; and Kahn et al., 1987, Tetrahedron Lett.28:1623-1626.

[0204] In still another embodiment of the invention, the multimers arein the form of branched networks (see, e.g., FIG. 7). Such networks areconveniently obtained through the use of multifunction linking moietiesthat permit more than two helical units to be attached to a simplelinking moiety. Thus, branched networks employ molecules having three,four or even more functional groups that are capable of covalentlyattaching to the N- and/or C-terminus of a peptide. Suitable linkingmoieties include, for example, amino acid residues having side chainsbearing hydroxyl, sulfanyl, amino, carboxyl, amide and/or esterfunctionalities, such as, for example, Ser (S), Thr (T), Cys (C), Tyr(Y), Asn (N), Gin (Q), Lys (K), Arg (R), Orn, Asp (D) and Glu (E); orother organic molecules containing such functional groups.

[0205] The helical segments attached to a single linking moiety need notbe attached via like termini. Indeed, in some embodiments the helicalsegments are attached to a single linking moiety so as to be arranged inan antiparallel fashion, i.e., some of the helices are attached viatheir N-termini, others via their C-termini.

[0206] The helical segments can be attached directly to the linkingmoiety, or may be spaced from the linking moiety by way of one or morebifunctional linkers (LL), as previously described.

[0207] Referring to FIGS. 7A and 7B, it can be seen that a branchednetwork can be described in terms of the number of “nodes” comprisingthe network, where each multifunctional linking moiety constitutes anode. In FIGS. 7A and 7B, helical segments (i.e., core peptides of theinvention) are illustrated as cylinders, and multifunctional linkingmoieties (or nodes) as circles (), where the number of lines emanatingfrom the circle indicates the “order” (or number of functional groups)of the multifunctional linking moiety.

[0208] The number of nodes in the network will generally depend on thetotal desired number of helical segments, and will typically be fromabout 1 to 2. Of course, it will be appreciated that for a given numberof desired helical segments, networks having higher order linkingmoieties will have fewer nodes. For example, referring to FIGS. 7A and7B, a tertiary-order network (i.e., a network having trifunctionallinking moieties) of seven helical units has three nodes (FIG. 7A),whereas a quaternary order network (i.e., a network havingtetrafunctional linking moieties) of seven helical units has only twonodes (FIG. 7B).

[0209] The networks may be of uniform order, i.e., networks in which allnodes are, for example, trifunctional or tetrafunctional linkingmoieties, or may be of mixed order, e.g., networks in which the nodesare mixtures of, for example, trifunctional and tetrafunctional linkingmoieties. Of course, it is to be understood that even in uniform ordernetworks the linking moieties need not be identical. A tertiary ordernetwork may employ, for example, two, three, four or even more differenttrifunctional linking moieties.

[0210] Like the linear multimers, the helical segments comprising thebranched network may be, but need not be, identical.

[0211] An example of such a mixed order branched network is illustratedin FIG. 7C. In FIG. 7C, helical segments (i.e., core peptides of theinvention) are illustrated as cylinders and multifunctional linkingmoieties as circles (), where the number of lines emanating from thecircle indicates the “order” (or number of functional groups) of themultifunctional linking moiety. Lines connecting helical segmentsrepresent bifunctional linkers LL, as previously described. Helicalsegments which comprise the branched networks may be tandem repeats ofcore peptides, as previously described.

[0212] In one illustrative embodiment, the branched networks of theinvention are described by the formula:

X—N_(ya)—X_((ya−1))N_(yb)—X_((yb−1)))_(p)  (III)

[0213] wherein:

[0214] each X is independently HHLL_(m)—HH_(n)LL_(m)—HH;

[0215] each HH is independently a core peptide of structure (I) or ananalogue or mutated, truncated, internally deleted or extended formthereof as described herein;

[0216] each LL is independently a bifunctional linker;

[0217] each m is independently an integer from 0 to 1;

[0218] each n is independently an integer from 0 to 8;

[0219] N_(ya) and N_(yb) are each independently a multifunctionallinking moiety where y_(a) and y_(b) represent the number of functionalgroups on N_(ya) and N_(yb), respectively;

[0220] each y_(a) or y_(b) is independently an integer from 3 to 8;

[0221] p is an integer from 0 to 7; and

[0222] each “—” independently designates a covalent bond.

[0223] In a preferred embodiment, the branched network comprises a“Lys-tree,” i.e., a network wherein the multifunctional linking moietyis one or more Lys (K) residues (see, e.g., FIG. 7D).

[0224] In one illustrative embodiment, the “Lys tree” branched networksof the invention are described by the formulae:

[0225] wherein:

[0226] each X is independently HHLL_(m)—HH_(n)LL_(m)—HH;

[0227] each HH is independently a core peptide or peptide analogue ofstructure (I) or a mutated, truncated, internally deleted or extendedform thereof as described herein;

[0228] each LL is independently a bifunctional linker;

[0229] each n is independently an integer from 0 to 8;

[0230] each m is independently an integer from 0 to 1;

[0231] R₁ is —OR or —NRR; and

[0232] each R is independently —H, (C₁-C₆) alkyl, (C₁-C₆) alkenyl,(C₁-C₆) alkynyl; (C₅-C₂₀) aryl (C₆-C₂₆) alkaryl, 5-20 memberedheteroaryl or 6-26 membered alkheteroaryl. 5.1.1. Analysis of Structureand Function

[0233] The structure and function of the core peptides or peptideanalogues of the invention, as well as ApoA-I agonists composed of suchcore peptides, including the multimeric forms described above, can beassayed in order to select active agonists or mimetics of ApoA-I. Forexample, the core peptides or peptide analogues can be assayed for theirability to form α-helices in the presence of lipids, to bind lipids, toform complexes with lipids, to activate LCAT, to promote cholesterolefflux, etc.

[0234] Methods and assays for analyzing the structure and/or function ofthe peptides are well-known in the art. Preferred methods are providedin the working examples, infra. For example, the circular dichroism (CD)and nuclear magnetic resonance (NMR) assays described in Section 7,infra, can be used to analyze the structure of the peptides or peptideanalogues—particularly the degree of helicity in the presence of lipids.The ability to bind lipids can be determined using the fluorescencespectroscopy assay described in Section 7, infra. The ability of thepeptides and/or peptide analogues to activate LCAT can be readilydetermined using the LCAT activation described in Section 8, infra. Thein vitro and in vivo assays described in Section 9, 10 and 11, infra,can be used to evaluate the half-life, distribution, cholesterol effluxand effects on RCT.

[0235] Generally, core peptides and/or peptide analogues according tothe invention which exhibit the properties listed in TABLE IV, infra,are considered to be active. TABLE IV PROPERTIES OF ACTIVE PEPTIDESPREFERRED PROPERTY RANGE RANGE % Helicity in the presence of ≧60% ≧80%lipids (Ri=30) (unblocked 22- amino acid residue peptides) % Helicity inthe presence of ≧40% ≧60% lipids (Ri=30) (unblocked 18- amino acidresidue peptides) % Helicity in the presence of ≧60% ≧80% lipids (Ri=30)(blocked 18-amino acid residue peptides and shorter peptides) LipidBinding (in the presence 0.5-10 μM of SUVs) peptide R_(i) =1-50 LCATactivation ≧38% ≧80%

[0236] R₁ is lipid:peptide molar ratio.

[0237] As illustrated in the working examples, infra, core peptideswhich exhibit a high degree of LCAT activation (≧38%) generally possesssignificant α-helical structure in the presence of lipidic smallunilamellar vesicles (SUVs) (≧60% helical structure in the case ofunblocked peptides containing 22 or more amino acid residues and blockedpeptides containing 18 or fewer amino acid residues; ≧40% helicalstructure in the case of unblocked peptides containing 18 or fewer aminoacids), and those peptides which exhibit little or no LCAT activationpossess little α-helical structure. However, in certain instances,peptides which exhibit significant helical structure in the presence oflipids do not effect significant LCAT.

[0238] Similarly, while core peptides that exhibit significant LCATactivation typically bind lipids, in certain instances peptides whichexhibit lipid binding do not effect significant LCAT activation.

[0239] As a consequence, it will be recognized by those of skill in theart that while the ability of the core peptides described herein to formα-helices (in the presence of lipids) and to bind lipids is critical foractivity, in many instances these properties may not be sufficient.Thus, in a preferred embodiment core peptides of the invention aresubjected to a series of screens to select for core peptides exhibitingsignificant pharmacological activity.

[0240] In a first step, a core peptide is screened for its ability toform an α-helix in the presence of lipids using the CD assay describedin Section 7, infra. Those peptides which are at least 40% helical(unblocked peptides containing 18 or fewer amino acids) or 60% helical(blocked peptides containing 18 or fewer amino acids; unblocked peptidescontaining 22 or more amino acids) in the presence of lipids (at a conc.of about 5 μM and a lipid:peptide molar ratio of about 30) are thenscreened for their ability to bind lipids using the fluorescence assaydescribed in Section 7, infra. Of course, only those core peptides whichcontain a fluorescent Trp (w) or Nal residue are screened for lipidbinding via fluorescence. However, for peptides which do not containfluorescent residues, binding to lipids is obvious when helicityincreases in the presence of lipids.

[0241] Core peptides which exhibit lipid binding in the presence of SUVs(0.5-10 μM peptide; lipid:peptide molar ratio in the range of 1 to 50)are then screened for pharmacological activity. Of course, thepharmacological activity screened for will depend upon the desired useof the ApoA-I agonists. In a preferred embodiment, the core peptides arescreened for their ability to activate LCAT, as peptides which activateLCAT are particularly useful in the methods described herein. Corepeptides which exhibit at least about 38% LCAT activation as comparedwith native human ApoA-I (as determined using the LCAT activation assaydescribed in Section 8, infra), are preferred, with core peptidesexhibiting 50%, 60%, 70%, 80% or even 90% or more being particularlypreferred.

5.1.2. Preferred Embodiments

[0242] The ApoA-I agonists of the invention can be further defined byway of preferred embodiments.

[0243] In one preferred embodiment, the ApoA-I agonists are 18 aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof.

[0244] In another preferred embodiment, the ApoA-I agonists are 18 aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich:

[0245] X₂ is Ala (A), Val (V) or Leu (L);

[0246] X₄ is Asp (D) or Glu (E);

[0247] X₇ is Arg (R), Lys (K) or Orn;

[0248] X₈ is Asp (D) or Glu (E);

[0249] X₁₁ is Glu (E) or Asn (N);

[0250] X₁₂ is Glu (E);

[0251] X₁₄ is Arg (R), Lys (K), Orn or Leu (L);

[0252] X₁₆ is Arg (R), Lys (K) or orn; and/or

[0253] X₁₈ is Arg (R), Lys (K) or Orn, and

[0254] X₁, X₃, X₅, X₆, X₉, X₁₀, X₁₃, X₁₅ and X₁₇ are as previouslydefined for structure (I).

[0255] In another preferred embodiment, the ApoA-I agonists are 18 aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich when X₁₁ is Asn (N) , X₁₄ is Leu (L) and when X₁₁ is other thanAsn (N), X₁₄ is other than Leu (L). Particularly preferred embodimentsaccording to this aspect of the invention are those peptides, or theN-terminal acylated and/or C-terminal amidated or esterified formsthereof, in which the various X_(n) in structure (I) are defined as inthe preceding paragraph. An exemplary particularly preferred embodimentaccording to this aspect of the invention is the peptide 209(PVLDLFRELLNELLQKLK; SEQ ID NO:209), and the N-terminal acylated and/orC-terminal amidated or esterified forms thereof.

[0256] In still another preferred embodiment, the ApoA-I agonists arealtered or mutated forms of the peptides according to structure (I), orthe N-terminal acylated and/or C-terminal amidated or esterified formsthereof, in which

[0257] X₁ is other than Asp (D);

[0258] X₉ is other than Gly (G);

[0259] X₁₀ is other than Gly (G)

[0260] X₁₂ is other than Leu (L); and

[0261] X₁₃ is other than Gly (G).

[0262] In still another preferred embodiment, the ApoA-I agonists areselected from the group of peptides set forth below: peptide 191PVLDLLRELLEELKQKLK* (SEQ ID NO:191); peptide 192 PVLDLFKELLEELKQKLK*(SEQ ID NO:192); peptide 193 PVLDLFRELLEELKQKLK* (SEQ ID NO:193);peptide 194 PVLELFRELLEELKQKLK* (SEQ ID NO:194); peptide 195PVLELFKELLEELKQKLK* (SEQ ID NO:195); peptide 196 PVLDLFRELLEELKNKLK*(SEQ ID NO:196); peptide 197 PLLDLFRELLEELKQKLK* (SEQ ID NO:197);peptide 198 GVLDLFRELLEELKQKLK* (SEQ ID NO:198); peptide 199PVLDLFRELWEELKQKLK* (SEQ ID NO:199); peptide 200 NVLDLFRELLEELKQKLK*(SEQ ID NO:200); peptide 201 PLLDLFKELLEELKQKLK* (SEQ ID NO:201);peptide 202 PALELFKDLLEELRQKLR* (SEQ ID NO:202); peptide 203AVLDLFRELLEELKQKLK* (SEQ ID NO:203); peptide 204 PVLDFFRELLEELKQKLK*(SEQ ID NO:204); peptide 205 PVLDLFREWLEELKQKLK* (SEQ ID NO:205);peptide 206 PLLELLKELLEELKQKLK* (SEQ ID NO:206); peptide 207PVLELLKELLEELKQKLK* (SEQ ID NO:207); peptide 208 PALELFKDLLEELRQRLK*(SEQ ID NO:208); peptide 209 PVLDLFRELLEELLQKLK (SEQ ID NO:209); peptide210 PVLDLFRELLEELKQKLK (SEQ ID NO:210); peptide 211 PVLDLFRELLEELOQOLO*(SEQ ID NO:211); peptide 212 PVLDLFOELLEELOQOLK* (SEQ ID NO:212);peptide 213 PALELFKDLLEEFRQRLK* (SEQ ID NO:213); peptide 214pVLDLFRELLEELKQKLK* (SEQ ID NO:214); peptide 215 PVLDLFRELLEEWKQKLK*(SEQ ID NO:215); peptide 229 PVLELFERLLEDLQKKLK (SEQ ID NO:229); peptide230 PVLDLFRELLEKLEQKLK (SEQ ID NO:230); peptide 231 PLLELFKELLEELKQKLK*(SEQ ID NO:231);

[0263] in either the N- and/or C-terminal blocked or unblocked forms.

[0264] In still another preferred embodiment, the ApoA-I agonists areselected from the group of peptides set forth below: peptide 191PVLDLLRELLEELKQKLK* (SEQ ID NO:191); peptide 192 PVLDLFKELLEELKQKLK*(SEQ ID NO:192); peptide 193 PVLDLFRELLEELKQKLK* (SEQ ID NO:193);peptide 194 PVLELFRELLEELKQKLK* (SEQ ID NO:194); peptide 195PVLELFKELLEELKQKLK* (SEQ ID NO:195); peptide 196 PVLDLFRELLEELKNKLK*(SEQ ID NO:196); peptide 197 PLLDLFRELLEELKQKLK* (SEQ ID NO:197);peptide 198 GVLDLFRELLEELKQKLK* (SEQ ID NO:198); peptide 199PVLDLFRELWEELKQKLK* (SEQ ID NO:199); peptide 200 NVLDLFRELLEELKQKLK*(SEQ ID NO:200); peptide 201 PLLDLFKELLEELKQKLK* (SEQ ID NO:201);peptide 202 PALELFKDLLEELRQKLR* (SEQ ID NO:202); peptide 203AVLDLFRELLEELKQKLK* (SEQ ID NO:203); peptide 204 PVLDFFRELLEELKQKLK*(SEQ ID NO:204); peptide 205 PVLDLFREWLEELKQKLK* (SEQ ID NO:205);peptide 206 PLLELLKELLEELKQKLK* (SEQ ID NO:206); peptide 207PVLELLKELLEELKQKLK* (SEQ ID NO:207); peptide 208 PALELFKDLLEELRQRLK*(SEQ ID NO:208); peptide 209 PVLDLFRELLNELLQKLK (SEQ ID NO:209); peptide210 PVLDLFRELLEELKQKLK (SEQ ID NO:210); peptide 211 PVLDLFRELLEELOQOLO*(SEQ ID NO:211); peptide 212 PVLDLFOELLEELOQOLK* (SEQ ID NO:212);peptide 213 PALELFKDLLEEFRQRLK* (SEQ ID NO:213); peptide 214pVLDLFRELLEELKQKLK* (SEQ ID NO:214); peptide 215 PVLDLFRELLEEWKQKLK*(SEQ ID NO:215);

[0265] in either the N- and/or C-terminal blocked or unblocked forms.

[0266] In yet another preferred embodiment, the ApoA-I agonists aremultimeric forms according to structures II, III and/or IV in which eachHH is independently a peptide according to structure (I) or anN-terminal acylated and/or C-terminal amidated or esterified formthereof, or any of the preferred peptides according to structure (I)described herein.

[0267] In yet another preferred embodiment, the core peptides thatcompose the ApoA-I agonists are not any of the following peptides:peptide 75: PVLDEFREKLNEELEALKQKLK (SEQ ID NO:75); peptide 94:PVLDEFREKLNEALEALKQKLK (SEQ ID NO:94); peptide 109:PVLDEFREKLNERLEALKQKLK (SEQ ID NO:109); peptide 237: LDDLELQKWAEAFNQLLKK(SEQ ID NO:237); peptide 238: EWLKAFYEKVLEKLKELF* (SEQ ID NO:238);peptide 241: DWFKAFYDKVFEKFKEFF (SEQ ID NO:241); peptide 242:GIKKFLGSIWKFIKAFVG (SEQ ID NO:242); peptide 243: DWFKAFYDKVAEKFKEAF (SEQID NO:243); peptide 244: DWLKAFYDKVAEKLKEAF (SEQ ID NO:244); peptide245: DWLKAFYDKVFEKFKEFF (SEQ ID NO:245); peptide 246: EWLEAFYKKVLEKLKELF(SEQ ID NO:246); peptide 247: DWFKAFYDKFFEKFKEFF (SEQ ID NO:247);peptide 248: EWLKAFYEKVLEKLKELF (SEQ ID NO:248); peptide 249:EWLKAEYEKVEEKLKELF* (SEQ ID NO:249); peptide 250: EWLKAEYEKVLEKLKELF*(SEQ ID NO:250); and peptide 251: EWLKAFYKKVLEKLKELF* (SEQ ID NO:251).

[0268] In a final preferred embodiment, the ApoA-I agonists are not anyof the peptides listed in TABLE X (Section 8.3, infra) which exhibit anLCAT activation activity of less than 38% as compared with native humanApoA-I.

5.2 Synthesis and Purification of the APoA-I Peptide Agonsits

[0269] The core peptides of the invention may be prepared usingvirtually any art-known technique for the preparation of peptides. Forexample, the peptides may be prepared using conventional step-wisesolution or solid phase peptide syntheses, or recombinant DNAtechniques.

5.2.1 Chemical Synthesis

[0270] Core peptides may be prepared using conventional step-wisesolution or solid phase synthesis (see, e.g., Chemical Approaches to theSynthesis of Peptides and Proteins, Williams et al., Eds., 1997, CRCPress, Boca Raton Fla., and references cited therein; Solid PhasePeptide Synthesis: A Practical Approach, Atherton & Sheppard, Eds.,1989, IRL Press, Oxford, England, and references cited therein).

[0271] Alternatively, the peptides of the invention may be prepared byway of segment condensation, as described, for example, in Liu et al.,1996, Tetrahedron Lett. 37(7):933-936; Baca, et al., 1995, J. Am. Chem.Soc. 117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein Res.45:209-216; Schnölzer and Kent, 1992, Science 256:221-225; Liu and Tam,1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam, 1994, Proc.Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li, 1988, Int. J.Peptide Protein Res. 31:322-334). Segment condensation is a particularlyuseful method for synthesizing embodiments containing internal glycineresidues. Other methods useful for synthesizing the peptides of theinvention are described in Nakagawa et al., 1985, J. Am. Chem. Soc.107:7087-7092.

[0272] ApoA-I agonists containing N- and/or C-terminal blocking groupscan be prepared using standard techniques of organic chemistry. Forexample, methods for acylating the N-terminus of a peptide or amidatingor esterifying the C-terminus of a peptide are well-known in the art.Modes of carrying other modifications at the N- and/or C-terminus willbe apparent to those of skill in the art, as will modes of protectingany side-chain functionalities as may be necessary to attach terminalblocking groups.

[0273] Pharmaceutically acceptable salts (counter ions) can beconveniently prepared by ion-exchange chromatography or other methods asare well known in the art.

[0274] Compounds of the invention which are in the form of tandemmultimers can be conveniently synthesized by adding the linker(s) to thepeptide chain at the appropriate step in the synthesis. Alternatively,the helical segments can be synthesized and each segment reacted withthe linker. Of course, the actual method of synthesis will depend on thecomposition of the linker. Suitable protecting schemes and chemistriesare well known, and will be apparent to those of skill in the art.

[0275] Compounds of the invention which are in the form of branchednetworks can be conveniently synthesized using the trimeric andtetrameric resins and chemistries described in Tam, 1988, Proc. Natl.Acad. Sci. USA 85:5409-5413 and Demoor et al., 1996, Eur. J. Biochem.239:74-84. Modifying the synthetic resins and strategies to synthesizebranched networks of higher or lower order, or which containcombinations of different core peptide helical segments, is well withinthe capabilities of those of skill in the art of peptide chemistryand/or organic chemistry.

[0276] Formation of disulfide linkages, if desired, is generallyconducted in the presence of mild oxidizing agents. Chemical oxidizingagents may be used, or the compounds may simply be exposed toatmospheric oxygen to effect these linkages. Various methods are knownin the art, including those described, for example, by Tam et al., 1979,Synthesis 955-957; Stewart et al., 1984, Solid Phase Peptide Synthesis,2d Ed., Pierce Chemical Company Rockford, IL; Ahmed et al., 1975, J.Biol. Chem. 250:8477-8482; and Pennington et al., 1991 Peptides 1990164-166, Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands. Anadditional alternative is described by Kamber et al., 1980, Helv. Chim.Acta 63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res. 26:92-97. Any of thesemethods may be used to form disulfide linkages in the peptides of theinvention.

5.2.2 Recombinant Synthesis

[0277] If the peptide is composed entirely of gene-encoded amino acids,or a portion of it is so composed, the peptide or the relevant portionmay also be synthesized using conventional recombinant geneticengineering techniques.

[0278] For recombinant production, a polynucleotide sequence encodingthe peptide is inserted into an appropriate expression vehicle, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation.The expression vehicle is then transfected into a suitable target cellwhich will express the peptide. Depending on the expression system used,the expressed peptide is then isolated by procedures well-established inthe art. Methods for recombinant protein and peptide production are wellknown in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y. each of which is incorporated byreference herein in its entirety.)

[0279] To increase efficiency of production, the polynucleotide can bedesigned to encode multiple units of the peptide separated by enzymaticcleavage sites—either homopolymers (repeating peptide units) orheteropolymers (different peptides strung together) can be engineered inthis way. The resulting polypeptide can be cleaved (e.g., by treatmentwith the appropriate enzyme) in order to recover the peptide units. Thiscan increase the yield of peptides driven by a single promoter. In apreferred embodiment, a polycistronic polynucleotide can be designed sothat a single mRNA is transcribed which encodes multiple peptides (i.e.,homopolymers or heteropolymers) each coding region operatively linked toa cap-independent translation control sequence; e.g., an internalribosome entry site (IRES). When used in appropriate viral expressionsystems, the translation of each peptide encoded by the mRNA is directedinternally in the transcript; e.g., by the IRES. Thus, the polycistronicconstruct directs the transcription of a single, large polycistronicmRNA which, in turn, directs the translation of multiple, individualpeptides. This approach eliminates the production and enzymaticprocessing of polyproteins and may significantly increase yield ofpeptide driven by a single promoter.

[0280] A variety of host-expression vector systems may be utilized toexpress the peptides described herein. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

[0281] The expression elements of the expression systems vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used inthe expression vector. For example, when cloning in bacterial systems,inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac(ptrp-lac hybrid promoter) and the like may be used; when cloning ininsect cell systems, promoters such as the baculovirus polyhedronpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll a/b binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter) may be used; when generating cell lines thatcontain multiple copies of expression product, SV40-, BPV- and EBV-basedvectors may be used with an appropriate selectable marker.

[0282] In cases where plant expression vectors are used, the expressionof sequences encoding the peptides of the invention may be driven by anyof a number of promoters. For example, viral promoters such as the 35SRNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature310:511-514), or the coat protein promoter of TMV (Takamatsu et al.,1987, EMBO J. 6:307-311) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3:1671-1680; Broglie et al., 1984, Science 224:838-843) or heat shockpromoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986,Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, e.g., Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, NY, SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9.

[0283] In one insect expression system that may be used to produce thepeptides of the invention, Autographa californica, nuclear polyhidrosisvirus (AcNPV) is used as a vector to express the foreign genes. Thevirus grows in Spodoptera frugiperda cells. A coding sequence may becloned into non-essential regions (for example the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed (e.g., see Smith et al., 1983, J. Virol. 46:584;Smith, U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Current Protocols in Molecular Biology, Vol. 2,Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.

[0284] In mammalian host cells, a number of viral based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5 Kpromoter may be used, (see, e.g., Mackett et al., 1982, Proc. Natl.Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol.49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

[0285] Other expression systems for producing the peptides of theinvention will be apparent to those having skill in the art.

5.2.3 Purification of Peptides

[0286] The peptides of the invention can be purified by art-knowntechniques such as reverse phase chromatography high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography and the like. The actual conditions used topurify a particular peptide will depend, in part, on synthesis strategyand on factors such as net charge, hydrophobicity, hydrophilicity, etc.,and will be apparent to those having skill in the art. Multimericbranched peptides can be purified, e.g., by ion exchange or sizeexclusion chromatography.

[0287] For affinity chromatography purification, any antibody whichspecifically binds the peptide may be used. For the production ofantibodies, various host animals, including but not limited to rabbits,mice, rats, etc., may be immunized by injection with a peptide. Thepeptide may be attached to a suitable carrier, such as BSA, by means ofa side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0288] Monoclonal antibodies to a peptide may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Kohler and Milstein,1975, Nature 256:495-497, or Kaprowski, U.S. Pat. No. 4,376,110 which isincorporated by reference herein; the human B-cell hybridoma technique)Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc.Natl. Acad. Sci. U.S.A. 80:2026-2030); and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96 (1985)). In addition, techniques developed for theproduction of “chimeric antibodies” Morrison et al., 1984, Proc. Natl.Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature312:604-608; Takeda et al., 1985, Nature 314:452-454, Boss, U.S. Pat.No. 4,816,397; Cabilly, U.S. Pat. No. 4,816,567; which are incorporatedby reference herein) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.Or “humanized” antibodies can be prepared (see, e.g., Queen, U.S. Pat.No. 5,585,089 which is incorporated by reference herein). Alternatively,techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce peptide-specific singlechain antibodies.

[0289] Antibody fragments which contain deletions of specific bindingsites may be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)₂ fragments, which can be producedby pepsin digestion of the antibody molecule and Fab fragments, whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor the peptide of interest.

[0290] The antibody or antibody fragment specific for the desiredpeptide can be attached, for example, to agarose, and theantibody-agarose complex is used in immunochromatography to purifypeptides of the invention. See, Scopes, 1984, Protein Purification:Principles and Practice, Springer-Verlag New York, Inc., NY,Livingstone, 1974, Methods In Enzymology: Immunoaffinity Chromatographyof Proteins 34:723-731.

5.3 Pharmaceutical Formulations and Methods of Treatment

[0291] The ApoA-I agonists of the invention can be used to treat anydisorder in animals, especially mammals including humans, for whichincreasing serum HDL concentration, activating LCAT, and promotingcholesterol efflux and RCT is beneficial. Such conditions include, butare not limited to hyperlipidemia, and especially hypercholesterolemia,and cardiovascular disease such as atherosclerosis (including treatmentand prevention of atherosclerosis); restenosis (e.g., preventing ortreating atherosclerotic plaques which develop as a consequence ofmedical procedures such as balloon angioplasty); and other disorders,such as endotoxemia, which often results in septic shock.

[0292] The ApoA-I agonists can be used alone or in combination therapywith other drugs used to treat the foregoing conditions. Such therapiesinclude, but are not limited to simultaneous or sequentialadministration of the drugs involved.

[0293] For example, in the treatment of hypercholesterolemia oratherosclerosis, the ApoA-I agonist formulations can be administeredwith any one or more of the cholesterol lowering therapies currently inuse; e.g., bile-acid resins, niacin, and/or statins. Such a combinedregimen may produce particularly beneficial therapeutic effects sinceeach drug acts on a different target in cholesterol synthesis andtransport; i.e., bile-acid resins affect cholesterol recycling, thechylomicron and LDL population; niacin primarily affects the VLDL andLDL population; the statins inhibit cholesterol synthesis, decreasingthe LDL population (and perhaps increasing LDL receptor expression);whereas the ApoA-I agonists affect RCT, increase HDL, increase LCATactivity and promote cholesterol efflux.

[0294] In another embodiment, the ApoA-I agonists may be used inconjunction with fibrates to treat hyperlipidemia, hypercholesterolemiaand/or cardiovascular disease such as atherosclerosis.

[0295] In yet another embodiment, the ApoA-I agonists of the inventioncan be used in combination with the anti-microbials andanti-inflammatory agents currently used to treat septic shock induced byendotoxin.

[0296] The ApoA-I agonists of the invention can be formulated aspeptides or as peptide-lipid complexes which can be administered tosubjects in a variety of ways to deliver the ApoA-l agonist to thecirculation. Exemplary formulations and treatment regimens are describedbelow.

5.3.1 ApoA-I Agonists and Peptide/Lipid Comples as the Active Ingredient

[0297] The ApoA-I agonist peptides can be synthesized or manufacturedusing any technique described in Section 5.2 and its subsections. Stablepreparations which have a long shelf life may be made by lyophilizingthe peptides—either to prepare bulk for reformulation, or to prepareindividual aliquots or dosage units which can be reconstituted byrehydration with sterile water or an appropriate sterile bufferedsolution prior to administration to a subject.

[0298] In certain embodiments, it may be preferred to formulate andadminister the ApoA-I agonist in a peptide-lipid complex. This approachhas several advantages since the complex should have an increasedhalf-life in the circulation, particularly when the complex has asimilar size and density to HDL, and especially the pre-β-1 or pre-β-2HDL populations. The peptide-lipid complexes can conveniently beprepared by any of a number of methods described below. Stablepreparations having a long shelf life may be made by lyophilization—theco-lyophilization procedure described below being the preferredapproach. The lyophilized peptide-lipid complexes can be used to preparebulk for pharmaceutical reformulation, or to prepare individual aliquotsor dosage units which can be reconstituted by rehydration with sterilewater or an appropriate buffered solution prior to administration to asubject.

[0299] A variety of methods well known to those skilled in the art canbe used to prepare the peptide-lipid vesicles or complexes. To this end,a number of available techniques for preparing liposomes orproteoliposomes may be used. For example, the peptide can be cosonicated(using a bath or probe sonicator) with appropriate lipids to formcomplexes. Alternatively the peptide can be combined with preformedlipid vesicles resulting in the spontaneous formation of peptide-lipidcomplexes. In yet another alternative, the peptide-lipid complexes canbe formed by a detergent dialysis method; e.g., a mixture of thepeptide, lipid and detergent is dialyzed to remove the detergent andreconstitute or form peptide-lipid complexes (e.g., see Jonas et al.,1986, Methods in Enzymol. 128:553-582).

[0300] While the foregoing approaches are feasible, each method presentsits own peculiar production problems in terms of cost, yield,reproducibility and safety. The applicants have developed a simplemethod for preparing peptide or protein-phospholipid complexes whichhave characteristics similar to HDL. This method can be used to preparethe ApoA-I peptide-lipid complexes, and has the following advantages:(1) Most or all of the included ingredients are used to form thedesigned complexes, thus avoiding waste of starting material which iscommon to the other methods. (2) Lyophilized compounds are formed whichare very stable during storage. The resulting complexes may bereconstituted immediately before use. (3) The resulting complexesusually need not be further purified after formation and before use. (4)Toxic compounds, including detergents such as cholate, are avoided.Moreover, the production method can be easily scaled up and is suitablefor GMP manufacture (i.e., in an endotoxin-free environment).

[0301] In accordance with the preferred method, the peptide and lipidare combined in a solvent system which co-solubilizes each ingredientand can be completely removed by lyophilization. To this end, solventpairs must be carefully selected to ensure co-solubility of both theamphipathic peptide and the lipid. In one embodiment, the protein(s) orpeptide(s) to be incorporated into the particles can be dissolved in anaqueous or organic solvent or mixture of solvents (solvent 1). The(phospho)lipid component is dissolved in an aqueous or organic solventor mixture of solvents (solvent 2) which is miscible with solvent 1, andthe two solutions are mixed. Alternatively, the peptide and lipid can beincorporated into a co-solvent system; i.e., a mixture of the misciblesolvents. A suitable proportion of peptide (protein) to lipids is firstdetermined empirically so that the resulting complexes possess theappropriate physical and chemical properties; i.e., usually (but notnecessarily) similar in size to HDL. The resulting mixture is frozen andlyophilized to dryness. Sometimes an additional solvent must be added tothe mixture to facilitate lyophilization. This lyophilized product canbe stored for long periods and will remain stable.

[0302] In the working examples describe infra, the peptide 210 (SEQ IDNO:210) and phospholipids were dissolved separately in methanol,combined, then mixed with xylene before lyophilization. The peptide andlipid can both be added to a mixture of the two solvents. Alternatively,a solution of the peptide dissolved in methanol can be mixed with asolution of lipid dissolved in xylene. Care should be taken to eliminatesalt from the solvent system in order to avoid salting out the peptide.The resulting solution containing the peptide and lipid cosolubilized inmethanol/xylene is lyophilized to form a powder.

[0303] The lyophilized product can be reconstituted in order to obtain asolution or suspension of the peptide-lipid complex. To this end, thelyophilized powder is rehydrated with an aqueous solution to a suitablevolume (often 5 mgs peptide/ml which is convenient for intravenousinjection). In a preferred embodiment the lyophilized powder isrehydrated with phosphate buffered saline or a physiological salinesolution. The mixture may have to be agitated or vortexed to facilitaterehydration, and in most cases, the reconstitution step should beconducted at a temperature equal to or greater than the phase transitiontemperature of the lipid component of the complexes. Within minutes, aclear preparation of reconstituted lipid-protein complexes results.

[0304] An aliquot of the resulting reconstituted preparation can becharacterized to confirm that the complexes in the preparation have thedesired size distribution; e.g., the size distribution of HDL. Gelfiltration chromatography can be used to this end. In the workingexamples described infra, a Pharmacia Superose 6 FPLC gel filtrationchromatography system was used. The buffer used contains 150 mM NaCl in50 mM phosphate buffer, pH 7.4. A typical sample volume is 20 to 200microliters of complexes containing 5 mgs peptide/ml. The column flowrate is 0.5 mls/min. A series of proteins of known molecular weight andStokes' diameter as well as human HDL are used as standards to calibratethe column. The proteins and lipoprotein complexes are monitored byabsorbance or scattering of light of wavelength 254 or 280 nm.

[0305] The ApoA-I agonists of the invention can be complexed with avariety of lipids, including saturated, unsaturated, natural andsynthetic lipids and/or phospholipids. Suitable lipids include, but arenot limited to, small alkyl chain phospholipids, eggphosphatidylcholine, soybean phosphatidylcholine,dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine,distearoylphosphatidylcholine1-myristoyl-2-palmitoylphosphatidylcholine,1-palmitoyl-2-myristoylphosphatidylcholine,1-palmitoyl-2-stearoylphosphatidylcholine,1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholinedioleophosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, sphingomyelin, sphingolipids,phosphatidylglycerol, diphosphatidylglycerol,dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol,distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol,dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid,dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,dipalmitoylphosphatidylserine, brain phosphatidylserine, brainsphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin,phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl etherglycolipids, and cholesterol and its derivatives.

[0306] The Applicants have discovered that when the ApoA-I agonists ofthe invention are complexed with sphingomyelin, all of the HDL of thepre-β-like particles is removed. Accordingly, in a preferred embodimentof the invention, the ApoA-I agonists are administered as a complex withsphingomyelin.

5.3.2 Methods of the Treatment

[0307] The ApoA-I peptide agonists or peptide-lipid complexes of theinvention may be administered by any suitable route that ensuresbioavailability in the circulation. This can best be achieved byparenteral routes of administration, including intravenous (IV),intramuscular (IM), intradermal, subcutaneous (SC) and intraperitoneal(IP) injections. However, other routes of administration may be used.For example, absorption through the gastrointestinal tract can beaccomplished by oral routes of administration (including but not limitedto ingestion, buccal and sublingual routes) provided appropriateformulations (e.g., enteric coatings) are used to avoid or minimizedegradation of the active ingredient, e.g., in the harsh environments ofthe oral mucosa, stomach and/or small intestine. Alternatively,administration via mucosal tissue such as vaginal and rectal modes ofadministration may be utilized to avoid or minimize degradation in thegastrointestinal tract. In yet another alternative, the formulations ofthe invention can be administered transcutaneously (e.g.,transdermally), or by inhalation. It will be appreciated that thepreferred route may vary with the condition, age and compliance of therecipient.

[0308] The actual dose of ApoA-I agonists or peptide-lipid complex usedwill vary with the route of administration, and should be adjusted toachieve circulating plasma concentrations of 100 mg/l to 2 g/l. Dataobtained in animal model systems described herein show that the ApoA-Iagonists of the invention associate with the HDL component, and have aprojected half-life in humans of about five days. Thus, in oneembodiment, the ApoA-I agonists can be administered by injection at adose between 0.5 mg/kg to 100 mg/kg once a week. In another embodiment,desirable serum levels may be maintained by continuous infusion or byintermittent infusion providing about 0.5 mg/kg/hr to 100 mg/kg/hr.

[0309] Toxicity and therapeutic efficacy of the various ApoA-I agonistscan be determined using standard pharmaceutical procedures in cellculture or experimental animals for determining the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. ApoA-I peptide agonists which exhibit largetherapeutic indices are preferred.

5.3.3 Pharmaceutical Formulations

[0310] The pharmaceutical formulation of the invention contain theApoA-I peptide agonist or the peptide-lipid complex as the activeingredient in a pharmaceutically acceptable carrier suitable foradministration and delivery in vivo. As the peptides may contain acidicand/or basic termini and/or side chains, the peptides can be included inthe formulations in either the form of free acids or bases, or in theform of pharmaceutically acceptable salts.

[0311] Injectable preparations include sterile suspensions, solutions oremulsions of the active ingredient in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives.

[0312] Alternatively, the injectable formulation may be provided inpowder form for reconstitution with a suitable vehicle, including butnot limited to sterile pyrogen free water, buffer, dextrose solution,etc., before use. To this end, the ApoA-I agonist may be lyophilized, orthe co-lyophilized peptide-lipid complex may be prepared. The storedpreparations can be supplied in unit dosage forms and reconstitutedprior to use in vivo.

[0313] For prolonged delivery, the active ingredient can be formulatedas a depot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt form of the ApoA-I agonist.

[0314] Alternatively, transdermal delivery systems manufactured as anadhesive disc or patch which slowly releases the active ingredient forpercutaneous absorption may be used. To this end, permeation enhancersmay be used to facilitate transdermal penetration of the activeingredient. A particular benefit may be achieved by incorporating theApoA-I agonists of the invention or the peptide-lipid complex into anitroglycerin patch for use in patients with ischemic heart disease andhypercholesterolemia.

[0315] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound.

[0316] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner. For rectal andvaginal routes of administration, the active ingredient may beformulated as solutions (for retention enemas) suppositories orointments.

[0317] For administration by inhalation, the active ingredient can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0318] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

5.4 Other Uses

[0319] The ApoA-I agonists of the invention can be used in assays invitro to measure serum HDL, e.g., for diagnostic purposes. Because theApoA-I agonists associate with the HDL component of serum, the agonistscan be used as “markers” for the HDL population. Moreover, the agonistscan be used as markers for the subpopulation of HDL that are effectivein RCT. To this end, the agonist can be added to or mixed with a patientserum sample; after an appropriate incubation time, the HDL componentcan be assayed by detecting the incorporated ApoA-I agonist. This can beaccomplished using labeled agonists (e., radiolabels, fluorescentlabels, enzyme labels, dyes, etc.), or by immunoassays using antibodies(or antibody fragments) specific for the agonist.

[0320] Alternatively, labeled agonist can be used in imaging procedures(e.g., CAT scans, MRI scans) to visualize the circulatory system, or tomonitor RCT, or to visualize accumulation of HDL at fatty streaks,atherosclerotic lesions, etc. (where the HDL should be active incholesterol efflux).

6. EXAMPLE Synthesis of Peptide Agonists of ApoA-I

[0321] The peptides described in TABLE X (Section 8.3, infra) weresynthesized and characterized as described in the subsections below. Thepeptides were also analyzed structurally and functionally as describedin Sections 7 and 8, infra.

6.1 Synthesis of Core Peptides

[0322] Peptides were synthesized on solid phase according to theMerrifield technique (Merrifield, 1969, J. Am. Chem. Soc. 85:2149-2154)using 0.25 mmol ρ-alkoxybenzylalcohol resin (HMP resin) (Wang, 1973, J.Am. Chem. Soc. 95:1328-1333) and Fmoc chemistry. All syntheses werecarried out on an Applied Biosystems ABI model 430A automated peptidesynthesizer (Perkin-Elmer, Foster City, Calif.). The salvation andactivation times used for each coupling cycle are shown in TABLE Vbelow: TABLE V SINGLE COUPLE ACTIVATOR CYCLES CYCLE DESIGNATEDDISSOLVING ACTIVATION TRANSFER NAME AMINO ACIDS SOLVENT TIME TIME TIMES*afmc 31 Asn(trt), ˜0.4 ml DCM ˜7 min. ˜51. min. 1 = 50 sec. His(trt),˜1.2 ml NMP 2 = 36 sec. Lys(Boc),Trp ˜1.0 ml HOBt/NMP afmc 32 Arg(Pmc),0.8 ml DCM ˜32 min. ˜51 min. 1 = 60 sec. Gln(trt),Aib 1.2 ml NMP 2 = 40sec. 1.0 ml HOBt/NMP afmc 33 Ala,Asp(OtBu), 0.4 ml DCM ˜4 min. ˜36.5 1 =38 sec. Glu(OtBu),Gly, 0.8 ml NMP min. 2 = 27 sec. Ile,Leu, 0.1 ml Met,Phe, Pro HOBt/NMP afmc 34 Val 0.4 ml DCM ˜4 min. ˜61.5 1 = 38 sec. 0.8ml NMP min. 2 = 27 sec. 0.1 ml HOBt/NMP

[0323] The resins were washed with NMP between each coupling step. Theprotocol for one synthesis cycle is shown below in TABLE VI: TABLE VICOUPLING PROTOCOL FOR ONE SYNTHESIS CYCLE OPERATION TIME (min.) 1.Deprotection (10% 20 piperdine in NMP) 2. Wash (NMP)  5 3. Couple (4equiv. Fmoc-amino 61 acid-HOBT ester in NMP, preactivated 50 min.) 4.Wash  3 5. Resin Sample (optional)  3 TOTAL 92

[0324] All amino acids except Fmoc-β-(1-naphthyl)alanine were coupled inthis manner. Fmoc-β-(1-naphthyl)alanine was coupled manually. For manualcoupling, 1 mmol Fmoc-β-(1-naphthyl)alanine and 1 mmol2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) were dissolved in 5 ml NMP and mixed with the peptide-resin.

[0325] Thereafter, 2 mmol of N-ethyldiisopropylamine were added, themixture shaken for 2 hours and the peptide-resin washed 6 times with 10ml NMP. The coupling efficiency was monitored using the Kaiser Test(Kaiser, 1970, Anal. Biochem. 34:59577), and the coupling repeated ifnecessary. After coupling of naphthylalanine, the remainder of thesynthesis was performed automatically as described above.

6.2 Synthesis of Peptide Amides

[0326] Where indicated in TABLE X (Section 8.3, infra), peptide amideswere synthesized using a Rink amide resin containing the Fmoc-Rink amidehandle 4-(2′,4′-dimethylphenyl)-Fmoc-phenoxymethyl (Rink, 1987,Tetrahedron Lett. 28:3787-3790) and the synthesis protocols described inSection 6.1, supra.

6.3 Synthesis of N-Terminal Acylated Peptides

[0327] Where indicated in TABLE X (Section 8.3, infra), N-terminalacylated forms of the peptides were prepared by exposing the resin-boundpeptide prepared as described in Section 6.1 or 6.2, supra, to anappropriate acylating agent.

[0328] For N-terminal acetylated peptides, 15 ml of acetic anhydridesolution (10% v/v in NMP) was added to each 1 g of resin-bound peptide,the mixture shaken for 5 min. and the resin recovered by filtration. Therecovered resin was washed three times with NMP (15 ml) and three timeswith ethanol (15 ml).

6.4 Cleavage and Deprotection

[0329] Following synthesis, the peptides described in Sections 6.1, 6.2and 6.3, supra, were cleaved from the resin and deprotected with acleavage solution containing 92.5% trifluroacetic acid (TFA)/3.75%anisole/3.75% dodecanthiol (v/v/v). To effect cleavage, 10 ml ofcleavage solution was added to 0.25 mmol peptide resin and stirred for1.5 hours at room temperature. The resin was removed via filtration andthe cleaved/deprotected peptide precipitated with diethyl ether, washedwith ether and dried in vacuo.

[0330] The cleavage cocktail for peptides containing Trp (W), as well asfor peptide amides, was composed of 86.5% TFA, 4.5% H₂0, 4.5%1,2-ethanedithiol, 4.5% anisole and 3% phenol.

6.5 Purification

[0331] The crude, cleaved peptides of Section 6.4 were purified byreverse phase HPLC. The purity of each peptide was confirmed bydifferent analytical techniques (analytical HPLC, capillaryelectrophoresis). Capillary electrophoreses were carried out on fusedsilica capillaries of 70 cm length and an internal diameter of 75 μm(Thermo Separation Products). The separations were performed at 25° C.,15 kV, run time 35 min., in two different buffer systems: Buffer 1 (20mM Na₂B₄O₇, pH 9.2) and Buffer 2 (10 mM Na₂HPO₄, pH 2.5). HPLCseparations were carried out on Nucleosil 7C18 or Nucleosil 7C4 columns(Macherey and Nagel, Germany), 250×21 mm, at a flow rate of 8 ml/min.The gradient elution was performed using a mixture of 0.1% TFA in water(Solvent A) and 0.1% TFA in acetonitrile (Solvent B). The gradients usedwere adjusted to meet the needs of each peptide.

6.6 Characterization

[0332] The mass and amino acid analysis of the purified peptidesdescribed in Section 6.5 were confirmed via mass spectrometry and aminoacid analysis, respectively, as described below. Edman degradation wasused for sequencing.

6.6.1 LC-MS

[0333] A standard commercially available triple stage quadruple massspectrometer (model TSQ 700; Finnigan MAT, San Jose Calif., USA) wasused for mass determination. A pneumatically assisted electrospray (ESI)interface was used for sample introduction to the atmospheric pressureionization source of the mass spectrometer. The interface sprayer wasoperated at a positive potential of 4.5 kV. The temperature of the steelcapillary was held at 200° C. whereas the manifold was at 70° C.Positive ions generated by this ion evaporation process entered theanalyzer of the mass spectrometer. The multiplier was adjusted to 1000V. The analyzer compartment of the mass spectrometer was at 4E-6. Allacquisitions were performed at resolution<1u.

[0334] Peptides were analyzed by direct infusion of the purifiedpeptides using an ABI (Applied Biosystems) microbore system consistingof a syringe pump (model 140B), an UV detector (model 785A) and anoven/injector (model 112A). The solvent system consisted of water(solvent A) and acetonitrile (solvent B), each containing 0.1% TFA.Peptides were infused using either a gradient or isocratic conditionsand eluted from an Aquapore C₁₈ column. The flow rate was typically 300μl/min. Concentration of each peptide was about 0.03 mg/ml, 20 μl ofwhich was injected (e.g., 30 pmol).

[0335] Full scan MS experiments were obtained by scanning quadruple 1from m/z 500-1500 in 4s. Data were acquired using an Alpha DEC stationand were processed using the software package provided by Finnigan MAT(BIOWORKS).

6.6.2 Amino Acid Analysis

[0336] Amino acid analysis was performed on an ABI (Applied Biosystems)420 Amino Acid Analyzer. This system consists of three modules: ahydrolysis and derivatisation instrument, a reverse-phase HPLC and adata system. Peptide sample were applied (3 times in triplicate) onporous glass slides and subsequently hydrolyzed under gas phaseconditions (155° C., 90 min.). After removal of the HCL, the resultingamino acids were converted to PTC-AA (Phenylthiocarbamoyl-amino acids)using PITC (Phenylisothiocyanate). After transfer to the HPC sample loopthe resulting mixtures were fractionated on an Aquapore C18 column usingthe gradient mode (Solvent A: 50 mmol ammonium acetate (NH₄Ac), pH 5.4,in water; Solvent B: 32 mmol of sodium acetate (NaOAc) in aqueousacetonitrile) under conditions of temperature control. The HPLC datawere processed by the software package provided by Applied Biosystems.Quantification was performed relative to a peptide standard delivered byApplied Biosystems.

6.7 Synthesis of Branched Networks

[0337] Tetrameric-core peptidyl resin and trimeric-core peptidyl resinare synthesized as described in Demoor et al., 1996, Eur. J. Biochem.239:74-84. The tetrameric and trimeric core matrix still linked to the4-methyl benzhydrylamine resin is then used as initial peptidyl-resinfor automated synthesis of core peptides as previously described.

[0338] Branched networks containing helical segments of different aminoacid compositions can be synthesized using orthogonal synthesis andprotecting strategies well known in the art.

7. EXAMPLE Structural and Lipid Binding Analysis of ApoA-I Peptides

[0339] The structural and lipid binding characteristics of the purifiedpeptides synthesized as described in Section 6, supra, were determinedby circular dichroism (CD), fluorescence spectroscopy and nuclearmagnetic resonance (NMR).

7.1 Circular Dichroism

[0340] This Example describes a preferred method for determining thedegree of helicity of the core peptides of the invention both free inbuffer and in the presence of lipids.

7.1.1 Experimental Method

[0341] Far UV circular dichroism spectra were recorded between 190 and260 nm (in 0.5 nm or 0.2 nm increments) with a AVIV62DS spectrometer(AVIV Associates, Lakewood, N.J., USA) equipped with a thermoelectriccell holder and sample changer. The instrument was calibrated with(+)-10-camphoric acid. Between one and three scans were collected foreach sample, using 10 cm, 5 cm, 1 cm and 0.1 cm path length quartzSuprasil cells, respectively, for peptide concentrations of 10⁻⁷ M to10⁻⁴ M. The bandwidth was fixed at 1.5 nm and the scan speed to 1s perwavelength step. The reported data are the mean of at least 2 or 3independent measurements.

[0342] After background substraction, spectra were converted to molarellipticity (θ) per residue in deg. cm⁻² dmol⁻¹. The peptideconcentration was determined by amino acid analysis and also byabsorption spectrometry on a Perkin Elmer Lambda 17 UV/Visiblespectrophotometer when the peptide contained a chromophore (tryptophane,dansyl, naphtylalanine).

[0343] CD spectra were obtained with free, unbound peptide (5 μM in 5 mMphosphate buffer, pH 7.4); with peptide-SUV complexes (20:1 EPC:Chol.,Ri=50); with peptide-micelle complexes(1-myristoyl-2-hydroxy-sn-glycero-3-phosphatidyl choline, Ri=100); andwith free, unbound peptide in the presence of 2,2,2-trifluoroethanol(TFE) (5 μM peptide, 90% vol TFE).

[0344] The SUVs were obtained by dispersing the lipids (10 mM, 20:1EPC:Chol., Avanti Polar Lipids, AL) in phosphate buffer (5 mM, pH 7.4)with bubbling N₂ for 5 min., followed by sonication (1.5 hr.) in a bathsonicator. The homogeneity of the preparation was checked by FPLC.

[0345] The micelles were obtained by dispersing the lipid (6 mM1-myristoyl-2-hydroxy-sn-glycero-3-phosphatidyl choline, Avanti PolarLipids, AL) in phosphate buffer (5 mM, pH 7.4) with bubbling N₂ for 5min., followed by vortexing.

[0346] To obtain the peptide-SUV complexes, SUVs were added to thepeptide (5 μM in 5 mM phosphate buffer, pH 7.4) at aphospholipid-peptide molar ratio (Ri) of 100.

[0347] To obtain the peptide-micelle complexes, micelles were added tothe peptide (5 μM in 5 mM phosphate buffer, pH 7.4) at a Ri of 100.

[0348] All spectra were recorded at 37° C. The stability of peptide 210(SEQ ID NO:210) as a function of temperature (both free in buffer and inmicelles) was determined by recording spectra at a series of differenttemperatures.

[0349] The degree of helicity of peptide 210 (SEQ ID NO:210) as afunction of concentration was also determined.

7.1.2 Helicity Determination

[0350] The degree of helicity of the peptides in the various conditionswas determined from the mean residue ellipticity at 222 nm (Chen et al.,1974, Biochemistry 13:3350-3359) or by comparing the CD spectra obtainedto reference spectra available on databases (16 helical referencespectra from Provencher & Glockner, 1981, Biochemistry 20:33-37;denatured protein reference spectra from Venyaminov et al., 1993, Anal.Biochem. 214:17-24) using the CONTIN curve-fitting algorithm version2DP, CD-1 pack (August 1982) (Provencher, 1982, Comput. Phys. Commun.27:213-227, 229-242). Acceptable fit was determined using thestatistical analysis methodology provided by the CONTIN algorithm. Theerror of all methods was ±5% helicity.

7.1.3 Results

[0351] The degree of helicity (%) of the free, unbound peptides (free),the peptide-SUV complexes (SUVs), the peptide-micelle complexes (mics)and the peptide-TFE solution (TFE) are reported in TABLE X, Section 8.3,infra.

[0352] Peptide 210 (SEQ ID NO:210) contains significant α-helicalstructure (63% helicity) in micelles. Moreover, the α-helical structureis completely stable over a temperature range of 5°-45° C. (data notshown). The helicity of peptide 210 (SEQ ID NO:210) also increases inthe presence of TFE, which is a solvent that, due to having asignficantly lower dielectric constant (∈=26.7) than water (∈=78.4),stabilizes α-helices and intrapeptide hydrogen bonds at concentrationsbetween 5-90% (v/v).

[0353] Referring to TABLE X, Section 8.3, infra, it can be seen thatthose peptides which exhibit a high degree of LCAT activation (≧38%)generally possess significant α-helical structure in the presence oflipids (≧60% ) helical structure in the case of unblocked peptidescontaining 22 or more amino acids or blocked peptides containing 18 orfewer amino acids; ≧40% helical structure in the case of unblockedpeptides containing 18 or fewer amino acids), whereas peptides whichexhibit little or no LCAT activation possess little α-helical structure.However, in some instances, peptides which contain significant α-helicalstructure in the presence of lipids do not exhibit significant LCATactivation. As a consequence, the ability of the core peptides of theinvention to adopt an α-helical structure in the presence of lipids isconsidered a critical feature of the core peptides of the invention, asthe ability to form an α-helix in the presence of lipids appears to be aprerequisite for LCAT activation.

7.2 Fluorescence Spectroscopy

[0354] The lipid binding properties of the peptides synthesized inSection 6, supra, were tested by fluorescence measurements with labeledpeptides, in the present case Tryptophane (Trp or W) or Naphtylalanine(Nal). The fluorescence spectra were recorded on a Fluoromax from Spex(Jobin-Yvon) equipped with a Xenon lamp of 150W, two monochromators(excitation and emission), a photomultiplier R-928 for detectionsensitive in the red up to 850 nm and a thermoelectric magnetic stirredcell holder. Quartz Suprasil cuvettes were used for measurements in themicromolar concentration range. A device of variable slits (from 0.4 to5 nm) allows modulation of the incident and emitted intensitiesaccording to the concentration of peptide used. The reported values arein general the average of between 2 to 4 spectra. The peptideconcentration is determined by absorption spectrometry on a Philips PU8800 using the absorption band of the Trp (∈_(280 nm)=5,550 M⁻¹ cm⁻¹ inTris buffer) or the Nal (∈_(224 nm)=92,770 M⁻¹ cm⁻¹ in methanol).

[0355] Fluorescence spectra of the peptides were recorded between 290 nmand 450 nm in Tris-HCl buffer (20 mM, pH=7.5), in the presence andabsence of lipidic vesicles. The small unilamellar vesicles were formedafter rehydration in buffer of the lyophilized phospholipids, dispersionand tip sonification under a N₂ stream. The lipids used were either EggPC/Chol. (20:1) or POPC/Chol. (20:1). The spectra were recorded at apeptide concentration of 2 μM and at a temperature of 37° C. Thefluorescence reference standard in the case of Trp wasN-acetyltryptophanylamide (NATA).

[0356] Lipid binding studies were done through progressive lipidicvesicle addition to the peptide in solution at 2 μM (slits:5 nm inexcitation and 1.5 nm in emission). Dilution effects were taken intoaccount for the fluorescence intensity determination. The lipidconcentrations were varied from 10 to 600 μM and the molar ratio oflipid to peptide (Ri) was varied from 5 to 300. The wavelength ofexcitation was set at 280 nm for both Trp and Nal.

7.2.1 Fluorescence Spectral Analysis

[0357] The data were directly recorded and treated by an IBM-PC linkedto the spectrofluorimeter through the DM3000F software from Spex. Thespectra were corrected by substraction of the solvent contribution andby application of a coefficient given by the constructor taking intoaccount the variation of the photomultiplier response versus thewavelength.

[0358] The fluorescence spectra of the peptides were characterized bythe wavelength at their maximum of fluorescence emission and by theirquantum yield compared to NATA in the case of peptides labeled with atryptophane. The process of binding to lipids was analyzed bycalculating the shift of the wavelength at the maximum of fluorescenceemission, (λ_(max)), and the variation of the relative fluorescenceintensity of emission versus the lipid concentration. The relativefluorescence intensity is defined as the following ratio:(I-I₀)_(λmax)/I_(0λmax). I and I₀ are both measured at the (λ_(max))corresponding to the initial free state of the peptide, i.e., withoutlipids. I is the intensity at a defined lipid to peptide ratio and I₀ isthe same parameter measured in absence of lipids. The absence of thesevariations is relevant of the absence of interactions of the peptideswith the lipids.

7.2.2 Results and Discussion

[0359] The lipid binding properties of peptide 199 (SEQ ID NO:199),which is similar in primary sequence to peptide 210 (SEQ ID NO:210)except that it contains a W (Trp) residue at position 10, are presentedin TABLE VII. TABLE VII BINDING PROPERTIES OF PEPTIDE 199 (SEQ IDNO:199) TO LIPIDIC VESICLES AS MEASURED BY FLUORESCENCE Lipid:PeptideMolar Ratio (Ri) I/I_(o) λ_(max) (nm)  0  0 348  5  8 344  10  8 339  3018 328  60 22 100 27 326 200 41 325

[0360] In buffer at a concentration of 2 μm, the maximum of thetryptophane fluorescence emission (λ_(max)) of peptide 199 (SEQ IDNO:199) is 348 nm. This corresponds to a tryptophane which is relativelyexposed to the aqueous environment when compared to NATA (λ max=350 nm).Peptide 199 (SEQ ID NO:199) binds very effectively to EPC/Chol (20:1)small unilamellar vesicles as demonstrated by the burying of thetryptophane (the wavelength for the tryptophane maximum fluorescenceemission shifts from 348 nm to 325 nm) and the high fluorescenceintensity exaltation (see Table VII). The burying of the tryptophaneresidue is maximal for a lipid to peptide molar ratio of about 100.

[0361] Other peptides which exhibited a high degree of helicity in thepresence of lipids (≧60% for unblocked peptides of ≧22 amino acids, orblocked peptides of ≦18 amino acids; ≧40% for unblocked peptides of ≦18amino acids) as measured by circular dichroism as disclosed in Section7.1, supra, also demonstrated good lipid binding. Of course, among allthe peptides selected by the circular dichroism screening, only the onesthat could be followed by fluorescence were tested for their lipidbinding properties.

7.3 Nuclear Magnetic Resonance (NMR)

[0362] This Example describes an NMR method for analyzing the structureof the core peptides of the invention.

7.3.1 NMR Sample Preparation

[0363] Samples were prepared by dissolving 5 mg of peptide in 90%H₂O/10% D₂O containing trace amounts of 2,2-Dimethyl-2-sila-5-pentanesulfonate (DSS) as an internal chemical shift reference. Some of thesamples contained trifluoroethanol (TFE) (expressed as % vol). The totalsample volume was 500 μl and the concentration of peptide wasapproximately 5 mM.

7.3.2 NMR Spectroscopy

[0364]¹H NMR spectra were acquired at 500 MHz using a Bruker DRX500spectrometer equipped with a B-VT2000 temperature control unit. One andtwo-dimensional experiments were recorded using standard pulsesequences. (Two Dimensional NMR Spectroscopy, Eds. W. R. Croasmun andRMK Carlson, 1994, VCH Publishers, New York, USA). Water suppression wasachieved with low power presaturation for 2 sec. Two-dimensionalexperiments were carried out in the phase sensitive mode using timeproportional phase incrementation (TPPI) and a spectral width of 6000 Hzin both dimensions. Typically, 40 scans were co-added for 400 t₁increments with 2048 data points. Data were processed using FELIX95software (Molecular Simulations) on an INDIGO2 workstation (SiliconGraphics). Data were zero-filled to give a 2K×2K data matrix andapodized by a 450 shifted squared sine-bell function.

7.3.3 NMR Assignment

[0365] Complete proton resonance assignments were obtained by applyingthe sequential assignment technique using DQFCOSY, TOCSY and NOESYspectra as described in the literature (Wuthrich, NMR of Proteins andNucleic Acids, 1986, John Wiley & Sons, New York, USA). Secondarychemical shifts were calculated for HN and Hα protons by subtracting thetabulated random coil chemical shifts (Wishart and Sykes, 1994, Method.Enz. 239:363-392) from the corresponding experimental values.

7.3.4 Results and Discussion

[0366] General Consideration. Amphipathic helical peptides tend toaggregate in aqueous solutions at the high concentrations necessary forNMR spectroscopy, making it difficult to obtain high resolution spectra.TFE is known to solubilize peptides, and in addition stabilizes helicalconformations of peptides having helical propensity. The findings fromNMR spectroscopy are demonstrated for peptide 210 (SEQ ID NO:210) as arepresentative example. The consensus 22-mer of Segrest (SEQ ID NO:75)was studied in comparison.

[0367] Secondary chemical shifts. Proton chemical shifts of amino acidsdepend both on the type of residue and on the local secondary structurewithin a peptide or protein (Szlagyi, 1995, Progress in Nuclear MagneticResonance Spectroscopy 27:325-443). Therefore, identification of regularsecondary structure is possible by comparing experimental shifts withtabulated values for random coil conformation.

[0368] Formation of an α-helix typically results in an up-field(negative) shift for the Hα resonance. Observation of an upfield Hαshift for several sequential residues is generally taken as evidence ofa helical structure. The Hα secondary shifts for peptide 210 (SEQ IDNO:210) in 25% TFE at 295 K show a significant negative shift forresidues 4 through 15 (FIG. 7A), demonstrating a highly helicalconformation. Small differences are observed in the Hα chemical shiftsof the consensus 22-mer (SEQ ID NO:75) compared to peptide 210 (SEQ IDNO:210).

[0369] The chemical shifts of amide hydrogens of amino acid residuesresiding in regions of α-helix are also shifted upfield with respect tothe chemical shifts observed for random coil. In addition, a periodicityof the HN shifts can be observed, and it reflects the period of thehelical turns. The amplitude of the shift variation along the sequenceis related to the amphipathicity of a helical peptide. A higherhydrophobic moment leads to a more pronounced oscillation (Zhou et al.,1992, J. Am. Chem. Soc. 114:4320-4326). The HN secondary shifts forpeptide 210 (SEQ ID NO:210) in 25% TFE at 295 K show an oscillatorybehavior in agreement with the amphipathic nature of the helix (FIG.7B).

[0370] The amino acid replacements lead to a more pronounced periodicityalong the entire sequence (FIG. 7B). The pattern clearly reflects thestronger amphipathic nature of peptide 210 (SEQ ID NO:210) as comparedto Segrest's consensus 22-mer (SEQ ID NO:75). The existence of 4-5helical turns can be discerned.

[0371] The secondary shift of an amide proton is influenced by thelength of the hydrogen bond to the carbonyl oxygen one turn away fromthe helix. Therefore, the periodicity of observed chemical shift valuesreflects different hydrogen bond lengths. This difference is associatedwith an overall curved helical shape of the helix backbone. Thehydrophobic residues are situated on the concave side. The secondaryshifts of peptide 210 (SEQ ID NO:210) indicate a curved α-helicalconformation.

8. EXAMPLE LCAT Activation Assay

[0372] The peptides synthesized as described in Section 6, supra, wereanalyzed in vitro for their ability to activate LCAT. In the LCAT assay,substrate vesicles (small unilamellar vesicles or “SUVs”) composed ofegg phophatidylcholine (EPC) or 1-Palmitoyl-2-oleyl-phosphatidylcholine(POPC) and radiolabelled cholesterol are preincubated with equivalentmasses either of peptide or ApoA-I (isolated from human plasma). Thereaction is initiated by addition of LCAT (purified from human plasma).Native ApoA-I, which was used as positive control, represents 100%activation activity. “Specific activity” (i.e., units of activity (LCATactivation)/unit of mass) of the peptides can be calculated as theconcentration of peptide that achieves maximum LCAT activation. Forexample, a series of concentrations of the peptide (e.g., a limitingdilution) can be assayed to determine the “specific activity” for thepeptide—the concentration which achieves maximal LCAT activation (i.e.,percentage conversion of cholesterol to cholesterol ester) at a specifictimepoint in the assay (e.g., 1 hr.). When plotting percentageconversion of cholesterol at, e.c., 1 hr., against the concentration ofpeptide used, the “specific activity” can be identified as theconcentration of peptide that achieves a plateau on the plotted curve.

8.1 Preparation of Substrate Vesicles

[0373] The vesicles used in the LCAT assay are SUVs composed of eggphosphatidylcholine (EPC) or 1-palmitoyl-2-oleyl-phosphatidylcholine(POPC) and cholesterol with a molar ratio of 20:1. To prepare a vesiclestock solution sufficient for 40 assays, 7.7 mg EPC (or 7.6 mg POPC; 10μmol), 78 μg (0.2 μmol) 4-¹⁴C-cholesterol, 116 μg cholesterol (0.3 μmol)are dissolved in 5 ml xylene and lyophilized. Thereafter 4 ml of assaybuffer is added to the dry powder and sonicated under nitrogenatmosphere at 4° C. Sonication conditions: Branson 250 sonicator, 10 mmtip, 6×5 minutes; Assay buffer: 10 mM Tris, 0.14 M NaCl, 1 mM EDTA, pH7.4). The sonicated mixture is centrifuged 6 times for 5 minutes eachtime at 14,000 rpm (16,000×g) to remove titanium particles. Theresulting clear solution is used for the enzyme assay.

8.2 Purification of LCAT

[0374] For the LCAT purification, dextran sulfate/Mg²⁺ treatment ofhuman plasma is used to obtain lipoprotein deficient serum (LPDS), whichis sequentially chromatographed on Phenylsepharose, Affigelblue,ConcanavalinA sepharose and anti-ApoA-I affinity chromatography, assummarized for a representative purification in TABLE IX, below: TABLEIX LCAT PURIFICATION Total Total Volume Protein Total Activity YieldPurification Fraction (ml) (mg) (nmol CE/mg*hr) (%) (fold) Plasma 55044550 63706 LPDS 500 31000 62620 98 1.4 Phenyl 210 363 51909 82 100sepharose Affigel 95 153 25092 39 115 blue ConA 43 36 11245 18 220sepharose Anti-A-I 120 3.5 5500 9 1109 Affinity

8.2.1 Preparation of LPDS

[0375] To prepare LPDS, 500 ml plasma is added to 50 ml dextran sulfate(MW=500000) solution. Stir 20 minutes. Centrifuge for 30 minutes at 3000rpm (16,000×g) at 4° C. Use supernatant (LPDS) for further purification(ca. 500 ml).

8.2.2 Phenylsepharose Chromatography

[0376] The following materials and conditions were used for thephenylsepharose chromatography. solid phase: Phenylsepharose fast flow,high subst. grade, Pharmacia column: XK26/40, gel bed height: 33 cm, V =ca. 175 ml flow rates: 200 ml/hr (sample) wash: 200 ml/hr (buffer)elution: 80 ml/hr (distilled water) buffer: 10 mM Tris, 140 mM NaCl, 1mM EDTA pH 7.4, 0.01% sodium azide.

[0377] Equilibrate the column in Tris-buffer, add 29 g NaCl to 500 mlLPDS and apply to the column. Wash with several volumes of Tris bufferuntil the absorption at 280 nm wavelength is approximately at thebaseline, then start the elution with distilled water. The fractionscontaining protein are pooled (pool size: 180 ml) and used forAffigelblue chromatography.

8.2.3 Affigelblue Chromatography

[0378] The Phenylsepharose pool is dialyzed overnight at 4° C. against20 mM Tris-HCl, pH 7.4, 0.01% sodium azide. The pool volume is reducedby ultrafiltration (Amicon YM30) to 50-60 ml and loaded on anAffigelblue column. solid phase: Affigelblue, Biorad, 153-7301 column,XK26/20, gel bed height: ca. 13 cm; column volume: approx. 70 ml. flowrates: loading: 15 ml/h wash: 50 ml/h

[0379] Equilibrate column in Tris-buffer. Apply Phenylsepharose pool tocolumn. Start in parallel to collect fractions. Wash with Tris-buffer.The pooled fractions (170 ml) were used for ConA chromatography.

8.2.4 ConA Chromatography

[0380] The Affigelblue pool was reduced via Amicon (YM30) to 30-40 mland dialyzed against ConA starting buffer (1 mM Tris HCl pH7.4; 1 mMMgCl₂, 1 mM MnCl₂, 1 mM CaCl₂, 0.01% Na-azide) overnight at 4° C. solidphase: ConA sepharose (Pharmacia) column: XK26/20, gel bed height: 14 cm(75 ml) flow rates: loading 40 ml/h washing (with starting buffer): 90ml/h elution: 50 ml/h, 0.2 M Methyl-α-D-mannoside in 1 mM Tris, pH 7.4.

[0381] The protein fractions of the mannoside elutions were collected(110 ml), and the volume was reduced by ultrafiltration (YM30) to 44 ml.The ConA pool was divided in 2 ml aliquots, which are stored at −20° C.

8.2.5 Anti-ApoA-I Affinity Chromatography

[0382] Anti-ApoA-I affinity chromatography was performed on Affigel-Hzmaterial (Biorad), to which the anti-ApoA-I abs have been coupledcovalently. column: XK16/20, V = 16 ml. The column was equilibrated withPBS pH 7.4. Two ml of the ConA pool was dialyzed for 2 hours against PBSbefore loading onto the column. flow rates: loading: 15 ml/hour washing(PBS) 40 ml/hour.

[0383] The pooled protein fractions (V=14 ml) are used for LCAT assays.

[0384] The column is regenerated with 0.1 M. Citrate buffer (pH 4.5) toelute bound A-I (100 ml), and immediately after this procedurereequilibrated with PBS.

8.3 Results

[0385] The results of the LCAT activation assay are presented in TABLEX, infra. TABLE X LCAT ACTIVATION EXHIBITED BY EXEMPLARY CORE PEPTIDESACTIVITY He (%) He (%) He (%) He (%) PEPTIDE AMINO ACID SEQUENCE (%)LCAT free mics SUVs TFE 1 (SEQ ID NO:1) PVLDLFRELLNELLEZLKQKLK 120% 7785 81 69  2 (SEQ ID NO:2) GVLDLFRELLNELLEALKQKLKK 105%  3 (SEQ ID NO:3)PVLDLFRELLNELLEWLKQKLK 98% 70 95 80 95  4 (SEQ ID NO:4)PVLDLFRELLNELLEALKQKLK 93% 80 95 97 94  5 (SEQ ID NO:5)PVLDLFRELLNELLEALKQKLKK 90%  6 (SEQ ID NO:6) PVLDLFRELLNEXLEALKQKLK 80%57 93 70 99  7 (SEQ ID NO:7) PVLDLFKELLNELLEALKQKLK 83% 77 89 85 73  8(SEQ ID NO:8) PVLDLFRELLNEGLEALKQKLK 83% 20 90 61 93  9 (SEQ ID NO:9)PVLDLFRELGNELLEALKQKLK 83%  10 (SEQ ID NO:10) PVLDLFRELLNELLEAZKQKLK 79%60 87 70 71  11 (SEQ ID NO:11) PVLDLFKELLQELLEALKQKLK 72%  12 (SEQ IDNO:12) PVLDLFRELLNELLEAGKQKLK 70%  13 (SEQ ID NO:13)GVLDLFRELLNEGLEALKQKLK 67%  14 (SEQ ID NO:14) PVLDLFRELLNELLEALOQOLO 61%70 96 80  15 (SEQ ID NO:15) PVLDLFRELWNELLEALKQKLK 60% 55 60 64 68  16(SEQ ID NO:16) PVLDLLRELLNELLEALKQKLK 59%  17 (SEQ ID NO:17)PVLELFKELLQELLEALKQKLK 59%  18 (SEQ ID NO:18) GVLDLFRELLNELLEALKQKLK 58% 19 (SEQ ID NO:19) pVLDLFRELLNEGLEALKQKLK 58%  20 (SEQ ID NO:20)PVLDLFREGLNELLEALKQKLK 57%  21 (SEQ ID NO:21) pVLDLFRELLNELLEALKQKLK 57% 22 (SEQ ID NO:22) PVLDLFRELLNELLEGLKQKLK 54%  23 (SEQ ID NO:23)PLLELFKELLQELLEALKQKLK 54%  24 (SEQ ID NO:24) PVLDLFRELLNELLEALQKKLK 53% 25 (SEQ ID NO:25) PVLDFFRELLNEXLEALKQKLK 51% 46 82 93  26 (SEQ IDNO:26) PVLDLFRELLNELLELLKQKLK 47%  27 (SEQ ID NO:27)PVLDLFRELLNELZEALKQKLK 44% 72 92 82 81  28 (SEQ ID NO:28)PVLDLFRELLNELWEALKQKLK 40% 82 98 90 81  29 (SEQ ID NO:29)AVLDLFRELLNELLEALKQKLK 39%  30 (SEQ ID NO:30) PVLDLFRELLNELLEALKQKLK 38%85 90 98 90  31 (SEQ ID NO:31) PVLDLFLELLNEXLEALKQKLK 34% 49 98 90  32(SEQ ID NO:32) XVLDLFRELLNELLEALKQKLK 33%  33 (SEQ ID NO:33)PVLDLFREKLNELLEALKQKLK 33%  34 (SEQ ID NO:34) PVLDZFRELLNELLEALKQKLK 32%58 67 68 62  35 (SEQ ID NO:35) PVLDWFRELLNELLEALKQKLK 31% 49 (sp) 59 61 36 (SEQ ID NO:36) PLLELLKELLQELLEALKQKLK 31% 95 100 95  37 (SEQ IDNO:37) PVLDLFREWLNELLEALKQKLK 29% 65 75 76 73  38 (SEQ ID NO:38)PVLDLFRELLNEXLEAWKQKLK 29% 25 49 21 49  39 (SEQ ID NO:39)PVLDLFRELLEELLKALKKKLK 25% 66 69 68 72  40 (SEQ ID NO:40)PVLDLFNELLRELLEALQKKLK 25% 66 84 79 77  41 (SEQ ID NO:41)PVLDLWRELLNEXLEALKQKLK 25% 53 73 85 69  42 (SEQ ID NO:42)PVLDEFREKLNEXWEALKQKLK 25% 15 74 27 76  43 (SEQ ID NO:43)PVLDEFREKLWEXLEALKQKLK 25%  44 (SEQ ID NO:44) pvldefreklneXlealkqklk 25%20 86  45 (SEQ ID NO:45) PVLDEFREKLNEXLEALKQKLK 24% 24 84 25 86  46 (SEQID NO:46) PVLDLFREKLNEXLEALKQKLK 23% 30 86 58 85  47 (SEQ ID NO:47)˜VLDLFRELLNEGLEALKQKLK 23%  48 (SEQ ID NO:48) pvLDLFRELLNELLEALKQKLK 22% 49 (SEQ ID NO:49) PVLDLFRNLLEKLLEALEQKLK 22% 57 65 52 57  50 (SEQ IDNO:50) PVLDLFRELLWEXLEALKQKLK 21% 68 84 89 76  51 (SEQ ID NO:51)PVLDLFWELLNEXLEALKQKLK 20% 63 82 81 73  52 (SEQ ID NO:52)PVWDEFREKLNEXLEALKQKLK 20% sp sp sp  53 (SEQ ID NO:53)VVLDLFRELLNELLEALKQKLK 19%  54 (SEQ ID NO:54) PVLDLFRELLNEWLEALKQKLK 19%76 71 84 78  55 (SEQ ID NO:55) P˜˜˜LFRELLNELLEALKQKLK 19% 38 72 78 75 56 (SEQ ID NO:56) PVLDLFRELLNELLEALKQKKK 18%  57 (SEQ ID NO:57)PVLDLFRNLLEELLKALEQKLK 18%  58 (SEQ ID NO:58) PVLDEFREKLNEXLEALKQKL˜ 18% 59 (SEQ ID NO:59) LVLDLFRELLNELLEALKQKLK 17%  60 (SEQ ID NO:60)PVLDLFRELLNELLEALKQ˜˜˜ 16% 39 83 66 84  61 (SEQ ID NO:61)PVLDEFRWKLNEXLEALKQKLK 16%  62 (SEQ ID NO:62) PVLDEWREKLNEXLEALKQKLK 16%15 85 43  63 (SEQ ID NO:63) PVLDFFREKLNEXLEALKQKLK 16%  64 (SEQ IDNO:64) PWLDEFREKLNEXLEALKQKLK 15%  65 (SEQ ID NO:65)˜VLDEFREKLNEXLEALKQKLK 15%  66 (SEQ ID NO:66) PVLDLFRNLLEELLEALQKKLK 15%64 82 66 70  67 (SEQ ID NO:67) ˜VLDLFRELLNELLEALKQKLK 14% 81 90 84 94 68 (SEQ ID NO:68) PVLDEFRELLKEXLEALKQKLK 14%  69 (SEQ ID NO:69)PVLDEFRKKLNEXLEALKQKLK 13%  70 (SEQ ID NO:70) PVLDEFRELLYEXLEALKQKLK 12%27 78 33 66  71 (SEQ ID NO:71) PVLDEFREKLNELXEALKQKLK 11%  72 (SEQ IDNO:72) PVLDLFRELLNEXLWALKQKLK 11% sp sp sp  73 (SEQ ID NO:73)PVLDEFWEKLNEXLEALKQKLK 10%  74 (SEQ ID NO:74) PVLDKFREKLNEXLEALKQKLK 10%75^(1/) (SEQ ID NO:75) PVLDEFREKLNEELEALKQKLK 10% 18 28 23 55  76 (SEQID NO:76) PVLDEFRELLFEXLEALKQKLK 9% 41 88 66  77 (SEQ ID NO:77)PVLDEFREKLNKXLEALKQKLK 9%  78 (SEQ ID NO:78) PVLDEFRDKLNEXLEALKQKLK  79(SEQ ID NO:79) PVLDEFRELLNELLEALKQKLK 9%  80 (SEQ ID NO:80)PVLDLFERLLNELLEALQKKLK 9%  81 (SEQ ID NO:81) PVLDEFREKLNWXLEALKQKLK  82(SEQ ID NO:82) ˜˜LDEFREKLNEXLEALKQKLK 8%  83 (SEQ ID NO:83)PVLDEFREKLNEXLEALWQKLK  84 (SEQ ID NO:84) PVLDEFREKLNELLEALKQKLK 7%  85(SEQ ID NO:85) P˜LDLFRELLNELLEALKQKLK 7% 58 61 64 69  86 (SEQ ID NO:86)PVLELFERLLDELLNALQKKLK 7%  87 (SEQ ID NO:87) pllellkellqellealkqklk 7%100 100 100  88 (SEQ ID NO:88) PVLDKFRELLNEXLEALKQKLK 7%  89 (SEQ IDNO:89) PVLDEFREKLNEXLWALKQKLK 6%  90 (SEQ ID NO:90)˜˜˜DEFREKLNEXLEALKQKLK 6%  91 (SEQ ID NO:91) PVLDEFRELLNEXLEALKQKLK 6%43 100 100  92 (SEQ ID NO:92) PVLDEFRELYNEXLEALKQKLK 5%  93 (SEQ IDNO:93) PVLDEFREKLNEXLKALKQKLK 5% 94^(2/) (SEQ ID NO:94)PVLDEFREKLNEALEALKQKLK 5% 18 70 27 63  95 (SEQ ID NO:95)PVLDLFRELLNLXLEALKQKLK 5% sp sp  96 (SEQ ID NO:96)pvldlfrellneXlealkqklk 5% 52 85 63 81  97 (SEQ ID NO:97)PVLDLFRELLNELLE˜˜˜˜˜˜˜ 4%  98 (SEQ ID NO:98) PVLDLFRELLNEELEALKQKLK 2% 99 (SEQ ID NO:99) KLKQKLAELLENLLERFLDLVP 2% 72 88 80 80 100 (SEQ IDNO:100) pvldlfrellnellealkqklk 2% 83 92 98 101 (SEQ ID NO:101)PVLDLFRELLNWXLEALKQKLK 2% sp sp 102 (SEQ ID NO:102)PVLDLFRELLNLXLEALKEKLK 2% sp 103 (SEQ ID NO:103) PVLDEFRELLNEELEALKQKLK1% 104 (SEQ ID NO:104) P˜˜˜˜˜˜˜LLNELLEALKQKLK 1% 21 49 29 55 105 (SEQ IDNO:105) PAADAFREAANEAAEAAKQKAK 1% 29 28 32 65 106 (SEQ ID NO:106)PVLDLFREKLNEELEALKQKLK 0% 107 (SEQ ID NO:107) .klkgklaellenllerfldlvp 0%sp sp 77 108 (SEQ ID NO:108) PVLDLFRWLLNEXLEALKQKLK 0% 28 55 54 109^(3/)(SEQ ID NO:109) PVLDEFREKLNERLEALKQKLK 0% 19 45 23 58 110 (SEQ IDNO:110) PVLDEFREKLNEXXEALKQKLK 0% 111 (SEQ ID NO:111)PVLDEFREKLWEXWEALKQKLK 0% 112 (SEQ ID NO:112) PVLDEFREKLNEXSEALKQKLK 0%113 (SEQ ID NO:113) PVLDEFREKLNEPLEALKQKLK 0% 6 22 114 (SEQ ID NO:114)PVLDEFREKLNEXMEALKQKLK 0% 115 (SEQ ID NO:115) PKLDEFREKLNEXLEALKQKLK 0%116 (SEQ ID NO:116) PHLDEFREKLNEXLEALKQKLK 0% 117 (SEQ ID NO:117)PELDEFREKLNEXLEALKQKLK 0% 118 (SEQ ID NO:118) PVLDEFREKLNEXLEALEQKLK 0%119 (SEQ ID NO:119) PVLDEFREKLNEELEAXKQKLK 0% 120 (SEQ ID NO:120)PVLDEFREKLNEELEXLKQKLK 0% 121 (SEQ ID NO:121) PVLDEFREKLNEELEALWQKLK 0%122 (SEQ ID NO:122) PVLDEFREKLNEELEWLKQKLK 0% 123 (SEQ ID NO:123)QVLDLFRELLNELLEALKQKLK 124 (SEQ ID NO:124) PVLDLFOELLNELLEALOQOLO 125(SEQ ID NO:125) NVLDLFRELLNELLEALKQKLK 126 (SEQ ID NO:126)PVLDLFRELLNELGEALKQKLK 127 (SEQ ID NO:127) PVLDLFRELLNELLELLKQKLK 47%128 (SEQ ID NO:128) PVLDLFRELLNELLEFLKQKLK 129 (SEQ ID NO:129)PVLELFNDLLRELLEALQKKLK 130 (SEQ ID NO:130) PVLELFNDLLRELLEALKQKLK 131(SEQ ID NO:131) PVLELFKELLNELLDALRQKLK 132 (SEQ ID NO:132)PVLDLFRELLENLLEALQKKLK 133 (SEQ ID NO:133) PVLELFERLLEDLLQALNKKLK 134(SEQ ID NQ:134) PVLELFERLLEDLLKALNQKLK 135 (SEQ ID NO:135)DVLDLFRELLNELLEALKQKLK 136 (SEQ ID NO:136) PALELFKDLLQELLEALKQKLK 137(SEQ ID NO:137) PVLDLFRELLNEGLEAZKQKLK 138 (SEQ ID NO:138)PVLDLFRELLNEGLEWLKQKLK 139 (SEQ ID NO:139) PVLDLFRELLWNEGLEALKQKLK 140(SEQ ID NO:140) PVLDLFRELLNEGLEALOQOLO 141 (SEQ ID NO:141)PVLDFFRELLNEGLEALKQKLK 142 (SEQ ID NO:142) PVLELFRELLNEGLEALKQKLK 143(SEQ ID NO:143) PVLDLFRELLNEGLEALKQKLK* 144 (SEQ ID NO:144)pVLELFENLLERLLDALQKKLK 111% 89 88 95 l45 (SEQ ID NO:145)GVLELFENLLERLLDALQKKLK 100% 55 51 58 146 (SEQ ID NO:146)PVLELFENLLERLLDALQKKLK 86% 97 100 100 95 147 (SEQ ID NO:147)PVLELFENLLERLFDALQKKLK 76% 148 (SEQ ID NO:148) PVLELFENLLERLGDALQKKLK75% 10 76 23 80 149 (SEQ ID NO:149) PVLELFENLWERLLDALQKKLK 63% 28 54 47150 (SEQ ID NO:150) PLLELFENLLERLLDALQKKLK 57% 151 (SEQ ID NO:151)PVLELFENLGERLLDALQKKLK 55% 152 (SEQ ID NO:152) PVFELFENLLERLLDALQKKLK50% 153 (SEQ ID NO:153) AVLELFENLLERLLDALQKKLK 49% 154 (SEQ ID NO:154)PVLELFENLLERGLDALQKKLK 39% 13 76 25 80 155 (SEQ ID NO:155)PVLELFLNLWERLLDALQKKLK 38% 156 (SEQ ID NO:156) PVLELFLNLLERLLDALQKKLK35% 157 (SEQ ID NO:157) PVLEFFENLLERLLDALQKKLK 30% 158 (SEQ ID NO:158)PVLELFLNLLERLLDWLQKKLK 30% 159 (SEQ ID NO:159) PVLDLFENLLERLLDALQKKLK28% 160 (SEQ ID NO:160) PVLELFENLLERLLDWLQKKLK 28% 65 73 75 61 161 (SEQID NO:161) PVLELFENLLERLLEALQKKLK 27% 162 (SEQ ID NO:162)PVLELFENWLERLLDALQKKLK 27% 68 83 81 163 (SEQ ID NO:163)PVLELFENLLERLWDALQKKLK 26% 27 53 55 164 (SEQ ID NO:164)PVLELFENLLERLLDAWQKKLK 24% 37 66 51 61 165 (SEQ ID NO:165)PVLELFENLLERLLDLLQKKLK 23% 166 (SEQ ID NO:166) PVLELFLNLLEKLLDALQKKLK22% 167 (SEQ ID NO:167) PVLELFENGLERLLDALQKKLK 18% 168 (SEQ ID NO:168)PVLELFEQLLEKLLDALQKKLK 17% 169 (SEQ ID NO:169) PVLELFENLLEKLLDALQKKLK17% 170 (SEQ ID NO:170) PVLELFENLLEOLLDALQOOLO 17% 171 (SEQ ID NO:171)PVLELFENLLEKLLDLLQKKLK 16% 172 (SEQ ID NO:172) PVLELFLNLLERLGDALQKKLK16% 173 (SEQ ID NO:173) PVLDLFDNLLDRLLDLLNKKLK 15% 174 (SEQ ID NO:174)pvlelfenllerlldalqkklk 13% 175 (SEQ ID NO:175) PVLELFENLLERLLELLNKKLK13% 176 (SEQ ID NO:176) PVLELWENLLERLLDALQKKLK 11% 177 (SEQ ID NO:177)GVLELFLNLLERLLDALQKKLK 10% 178 (SEQ ID NO:178) PVLELFDNLLEKLLEALQKKLR 9%179 (SEQ ID NO:179) PVLELFDNLLERLLDALQKKLK 8% 180 (SEQ ID NO:180)PVLELFDNLLDKLLDALQKKLR 8% 181 (SEQ ID NO:181) PVLELFENLLERWLDALQKKLK 8%182 (SEQ ID NO:182) PVLELFENLLEKLLEALQKKLK 7% 183 (SEQ ID NO:183)PLLELFENLLEKLLDALQKKLK 6% 184 (SEQ ID NO:184) PVLELFLNLLERLLDAWQKKLK 4%185 (SEQ ID NO:185) PVLELFENLLERLLDALQOOLO 3% 186 (SEQ ID NO:186)PVLELFEQLLERLLDALQKKLK 187 (SEQ ID NO:187) PVLELFENLLERLLDALNKKLK 188(SEQ ID NO:188) PVLELFENLLDRLLDALQKKLK 189 (SEQ ID NO:189)DVLELFENLLERLLDALQKKLK 190 (SEQ ID NO:190) PVLEFWDNLLDKLLDALQKKLR 191(SEQ ID NO:191) PVLDLLRELLEELKQKLK* 100% 192 (SEQ ID NO:192)PVLDLFKELLEELKQKLK* 100% 36 56 193 (SEQ ID NO:193) PVLDLFRELLEELKQKLK*96% 34 88 87 87 194 (SEQ ID NO:194) PVLELFRELLEELKQKLK* 88% 38 93 93 195(SEQ ID NO:195) PVLELFKELLEELKQKLK* 87% 196 (SEQ ID NO:196)PVLDLFRELLEELKNKLK* 81% 197 (SEQ ID NO:197) PLLDLFRELLEELKQKLK* 81% 4370 69 198 (SEQ ID NO:198) GVLDLFRELLEELKQKLK* 80% 199 (SEQ ID NO:199)PVLDLFRELWEELKQKLK* 76% 35 77 80 79 200 (SEQ ID NO:200)NVLDLFRELLEELKQKLK* 75% 201 (SEQ ID NO:201) PLLDLFKELLEELKQKLK* 74% 202(SEQ ID NO:202) PALELFKDLLEELRQKLR* 70% 203 (SEQ ID NO:203)AVLDLFRELLEELKQKLK* 66% 204 (SEQ ID NO:204) PVLDFFRELLEELKQKLK* 63% 205(SEQ ID NO:205) PVLDLFREWLEELKQKLK* 60% 206 (SEQ ID NO:206)PLLELLKELLEELKQKLK* 57% 207 (SEQ ID NO:207) PVLELLKELLEELKQKLK* 50% 208(SEQ ID NO:208) PALELFKDLLEELRQRLK* 48% 209 (SEQ ID NO:209)PVLDLFRELLNELLQKLK 47% 54 71 67 62 210 (SEQ ID NO:210)PVLDLFRELLEELKQKLK 46% 20 63 37 53 211 (SEQ ID NO:211)PVLDLFRELLEELOQOLO* 45% 212 (SEQ ID NO:212) PVLDLFOELLEELOQOLK* 43% 213(SEQ ID NO:213) PALELFKDLLEEFRQRLK* 42% 214 (SEQ ID NO:214)pVLDLFRELLEELKQKLK* 39% 215 (SEQ ID NO:215) PVLDLFRELLEEWKQKLK* 38% 2863 53 68 216 (SEQ ID NO:216) PVLELFKELLEELKQKLK 35% 217 (SEQ ID NO:217)PVLDLFRELLELLKQKLK 30% 52 78 76 70 218 (SEQ ID NO:218)PVLDLFRELLNELLQKLK* 29% 219 (SEQ ID NO:219) PVLDLFRELLNELWQKLK 24% 220(SEQ ID NO:220) PVLDLFRELLEELQKKLK 22% 27 64 54 64 221 (SEQ ID NO:221)DVLDLFRELLEELKQKLK* 12% 222 (SEQ ID NO:222) PVLDAFRELLEALLQLKK 8% 223(SEQ ID NO:223) PVLDAFRELLEALAQLKK 8% 21 56 23 51 224 (SEQ ID NO:224)PVLDLFREGWEELKQKLK 8% 225 (SEQ ID NO:225) PVLDAFRELAEALAQLKK 1% 226 (SEQID NO:226) PVLDAFRELGEALLQLKK 1% 227 (SEQ ID NO:227) PVLDLFRELGEELKQKLK*228 (SEQ ID NO:228) PVLDLFREGLEELKQKLK* 0% 229 (SEQ ID NO:229)PVLDLFRELLEEGKQKLK* 0% 230 (SEQ ID NO:230) PVLELFERLLEDLQKKLK 231 (SEQID NO:231) PVLDLFRELLEKLEQKLK 232 (SEQ ID NO:232) PLLELFKELLEELKQKLK*237^(4/) (SEQ ID NO:237) LDDLLQKWAEAFNQLLKK 11% 30 66 45 238^(5/) (SEQID NO:238) EWLKAFYEKVLEKLKELF* 19% 49 72 60 58 239^(6/) (SEQ ID NO:239)EWLEAFYKKVLEKLKELF* 11% 44 49 sp 240 (SEQ ID NO:240) DWLKAFYDKVAEKLKEAF*10% 16 68 59 57 241 (SEQ ID NO:241) DWFKAFYDKVFEKFKEFF 8% 242^(7/) (SEQID NO:242) GIKKFLGSIWKFIKAFVG 7% 243 (SEQ ID NO:243) DWFKAFYDKVAEKFKEAF5% 10 64 50 244^(8/) (SEQ ID NO:244) DWLKAFYDKVAEKLKEAF 5% 9 40 13 48245 (SEQ ID NO:245) DWLKAFYDKVFEKFKEFF 4% 38 77 70 sp 246^(9/) (SEQ IDNO:246) EWLEAFYKKVLEKLKELF 4% 18 44 47 247 (SEQ ID NO:247)DWFKAFYDKFFEKFKEFF 3% 248^(10/) (SEQ ID NO:248) EWLKAFYEKVLEKLKELF 3% 1845 13 249^(11/) (SEQ ID NO:249) EWLKAEYEKVEEKLKELF* 250^(12/) (SEQ IDNO:250) EWLKAEYEKVLEKLKELF* 251^(13/) (SEQ ID NO:251)EWLKAFYKKVLEKLKELF* 252 (SEQ ID NO:252) PVLDLFRELLEQKLK* 253 (SEQ IDNO:253) PVLDLFRELLEELKQK* 254 (SEQ ID NO:254) PVLDLFRELLEKLKQK* 255 (SEQID NO:255) PVLDLFRELLEKLQK* 256 (SEQ ID NO:256) PVLDLFRELLEALKQK* 257(SEQ ID NO:257) PVLDLFENLLERLKQK* 258 (SEQ ID NO:258) PVLDLFRELLNELKQK*. (3) :580-588).

[0386] In TABLE X, * indicates peptides that are N-terminal acetylatedand C-terminal amidated; ↑ indicates peptides that are N-terminaldansylated; sp indicates peptides that exhibited solubility problemsunder the experimental conditions; X is Aib; Z is Nal; O is Orn; He (%)designates percent helicity; mics designates micelles; and ˜ indicatesdeleted amino acids.

9. EXAMPLE Pharmacokinetics of the ApoA-I Agonists

[0387] The following experiments can be used to demonstrate that theApoA-I agonists are stable in the circulation and associate with the HDLcomponent of plasma.

9.1. Synthesis of Radiolabelled Peptide

[0388] Radiolabelled peptides are synthesized by coupling a ¹⁴C-labeledamino acid as the N-terminal amino acid. The synthesis is carried outaccording to L. Lapatsanis, Synthesis, 1983, 671-173. Briefly, 250 μM ofunlabeled N-terminal amino acid is dissolved in 225 μl of a 9% Na₂CO₃solution and added to a solution (9% Na₂CO₃) of 9.25 MBq (250 μM)¹⁴C-labeled N-terminal amino acid. The liquid is cooled down to 0° C.,mixed with 600 μM (202 mg) 9-fluorenylmethyl-N-succinimidylcarbonate(Fmoc-OSu) in 0.75 ml DMF and shaken at room temperature for 4 hr.Thereafter the mixture is extracted with Diethylether (2×5 ml) andchloroform (1×5 ml), the remaining aqueous phase is acidified with 30%HCl and extracted with chloroform (5×8 ml). The organic phase is driedover Na₂SO₄, filtered off and the volume was reduced under nitrogen flowto 5 ml. The purity is estimated by TLC (CHCl₃:MeOH:Hac, 9:1:0.1 v/v/v,stationary phase RPTLC silicagel 60, Merck, Germany).

[0389] The chloroform solution containing ¹⁴C-labeled Fmoc amino acid isused directly for peptide synthesis. A peptide resin containing aminoacids 2-22 is synthesized automatically as described in Section 6. Thesequence of the peptide is determined by Edman degradation. The couplingis performed as described in Section 6.1.

9.2. Pharmacokinetics in Mice

[0390] In each experiment, 2.5 mg/kg radiolabelled peptide is injectedintraperitoneally into mice which are fed normal mouse chow or theatherogenic Thomas-Harcroft modified diet (resulting in severelyelevated VLDL and IDL cholesterol). Blood samples are taken at multipletime intervals for assessment of radioactivity in plasma.

9.3. Stability in Human Serum

[0391] The stability of the ApoA-I agonists of the invention in humanserum is demonstrated as described below.

9.3.1. Experimental Methods

[0392] 100 μg of ¹⁴C-labeled peptide (prepared as described in Section9.1, supra), is mixed with 2 ml of fresh human plasma (at 37° C.) anddelipidated either immediately (control sample) or after 8 days ofincubation at 37° C. (test sample). Delipidation is carried out byextracting the lipids with an equal volume of 2:1 (v/v)chloroform:methanol.

[0393] The samples are loaded onto a reverse-phase C₁₈ HPLC column andeluted with a linear gradient (25-58w over 33 min.) of acetonitrile(containing 0.1% TFA). Elution profiles are followed by absorbance (220nm) and radioactivity.

9.4. Formation of Pre-β Like Particles

[0394] The ability of the ApoA-I agonists of the invention to formpre-β-like particles is demonstrated as described below.

9.4.1. Experimental Method

[0395] Human HDL is isolated by KBr density ultra centrifugation atdensity d=1.21 g/ml to obtain top fraction followed by Superose 6 gelfiltration chromatography to separate HDL from other lipoproteins.Isolated HDL is adjusted to a final concentration of 1.0 mg/ml withphysiological saline based on protein content determined by Bradfordprotein assay. An aliquot of 300 μl is removed from the isolated HDLpreparation and incubated with 100 μl ¹⁴C-labeled peptide for two hoursat 37° C. Five separate incubations are analyzed including a blankcontaining 100 μl physiological saline and four dilutions of ¹⁴C-labeledpeptide: (i) 0.20 μg/gl peptide:HDL, ratio=1:15; (ii) 0.30 μg/glpeptide:HDL, ratio=1:10; (iii) 0.60 μg/gl peptide:HDL, ratio=1:5; and(iv) 1.00 μg/μl peptide:HDL, ratio=1:3. Following the two hourincubation, a 200 μl aliquot of the sample (total volume=400 μl) isloaded onto a Superose 6 gel filtration column for lipoproteinseparation and analysis, and 100 μl is used to determine totalradioactivity loaded onto the column.

9.5. Association of Apo-A-I Agonists with Human Lipoproteins 9.5.1.Experimental Methods

[0396] The ability of the ApoA-I agonists of the invention to associatewith human lipoprotein fractions is determined by incubating ¹⁴C-labeledpeptide with each lipoprotein class (HDL, LDL and VLDL) and a mixture ofthe different lipoprotein classes.

[0397] HDL, LDL and VLDL are isolated by KBr density gradientultracentrifugation at d=1.21 g/ml and purified by FPLC on a Superose 6Bcolumn size exclusion column (chromatography is carried out at a flowrate of 0.7 ml/min. and a running buffer of 10 mM Tris (pH 8), 115 mMNaCl, 2 mM EDTA and 0.01% NaN₃). ¹⁴C-labeled peptide is incubated withHDL, LDL and VLDL at a peptide:phospholipid ratio of 1:5 (mass ratio)for 2 h at 37° C. The required amount of lipoprotein (volumes based onamount needed to yield 1000 μg) is mixed with 0.2 ml of peptide stocksolution (1 mg/ml) and the solution is brought up to 2.2 ml using 0.9%of NaCl.

[0398] After incubating for 2 hr. at 37° C., an aliquot (0.1 ml) isremoved for liquid scintillation counting to determine the totalradioactivity, the density of the remaining incubation mixture isadjusted to 1.21 g/ml with KBr, and the samples are centrifuged at100,000 rpm (300,000 g) for 24 hours at 4° C. in a TLA 100.3 rotor usinga Beckman tabletop ultracentrifuge. The resulting supernatant isfractionated by removing 0.3 ml aliquots from the top of each sample fora total of 5 fractions, and 0.05 ml of each fraction is used for liquidscintillation counting. The top two fractions contain the floatinglipoproteins, the other fractions (3-5) correspond to proteins/peptidesin solution.

9.6. The ApoA-I Agonists of the Invention Selectively Bind HDL Lipids inHuman Plasma 9.6.1. Experimental Method

[0399] To demonstrate that the ApoA-I agonists of the inventionselectively bind HDL proteins in human plasma, 2 ml of human plasma isincubated with 20, 40, 60, 80, and 100 μg of ¹⁴C-labeled peptide for 2hr. at 37° C. The lipoproteins are separated by adjusting the density to1.21 g/ml and centrifugation in a TLA 100.3 rotor at 100,000 rpm(300,000 g) for 36 hr. at 4° C. The top 900 μl (in 300 μl fractions) istaken for analysis. 50 μl from each 300 μl fraction is counted forradioactivity and 200 μl from each fraction is analyzed by FPLC(Superose 6/Superose 12 combination column).

10. EXAMPLE The ApoA-I Agonists Promote Cholesterol Efflux

[0400] To demonstrate that the ApoA-I agonists of the invention promotecholesterol efflux, HepG2 hepatoma cells are plated into 6-well culturedishes and grown to confluence. Cells are labeled with ³H-cholesterol bydrying the cholesterol, then adding 1% bovine serum albumin (BSA) inphosphate buffered saline (PBS), sonicating the solution, and adding 0.2ml of this labeling solution and 1.8 ml growth medium to the cells, sothat each well contains 2 μCi of radioactivity. Cells are incubated for24 hr. with the labeling medium.

[0401] Peptide (or protein):DMPC complexes are prepared at a 1:2 peptide(or protein):DMPC ratio (w:w). To prepare the complexes, peptide ornative human ApoA-I protein is added to a DMPC solution in PBS andincubated at room temperature overnight, by which time the solution willclarify. Peptide or protein concentration in the final solution is about1 mg/ml.

[0402] Labeling media is removed from the cells and the cells are washedwith PBS prior to addition of complexes. 1.6 ml of growth medium isadded to each well, followed by peptide (or protein) :DMPC complex andsufficient PBS to bring the final volume to 2 ml per well. The finalpeptide or ApoA-I concentrations are about 1, 2.5, 5, 7.5 and 25 μg/mlmedium. After 24 hours of incubation at 37° C., the medium is removed,and the cells are washed with 2 ml of 1% BSA/PBS, followed by 2 washeswith 2 ml each of PBS. The amount of ³H-cholesterol effluxed into themedium is determined by liquid scintillation counting.

11. EXAMPLE Use of the ApoA-I Agonists in Animal Model Systems

[0403] The efficacy of the ApoA-I agonists of the invention can bedemonstrated in rabbits using the protocols below.

11.1. Preparation of the Phospholipid/Peptide Complexes

[0404] Small discoidal particles consisting of phospholipid (DPPC) andpeptide are prepared following the cholate dialysis method. Thephospholipid is dissolved in chloroform and dried under a stream ofnitrogen. The peptide is dissolved in buffer (saline) at a concentrationof 1-2 mg/ml. The lipid film is redissolved in buffer containing cholate(43° C.) and the peptide solution is added at a 3:1 phospholipid/peptideratio. The mixture is incubated overnight at 43° C. and then dialyzed at43° C. (24 hr.), room temperature (24 hr.) and 4° C. (24 hr.), withthree changes of buffer (large volumes) at temperature point. Thecomplexes are filter sterilized (0.22μ) for injection and storage at 4°C.

11.2. Isolation and Characterization of the Peptide/PhospholipidParticles

[0405] The particles are separated on a gel filtration column (Superose6 HR). The position of the peak containing the particles is identifiedby measuring the phospholipid concentration in each fraction. From theelution volume, the Stokes radius can be determined. The concentrationof peptide in the complex is determined by determining the phenylalaninecontent (by HPLC) following a 16 hr. acid hydrolysis.

11.3. Injection in the Rabbit

[0406] Male New Zealand White rabbits (2.5-3 kg) are injectedintravenously with a dose of phospholipid/peptide complex (5-10 mg/kgbodyweight peptide or 10 mg/kg bodyweight ApoA-I (control)), expressedas peptide or protein content) in a single bolus injection not exceeding10-15 ml. The animals are slightly sedated before the manipulations.Blood samples (collected on EDTA) are taken before and 5, 15, 30, 60,240 and 1440 minutes after injection. The hematocrit (Hct) is determinedfor each sample. Samples are aliquoted and stored at −20° C. beforeanalysis.

11.4. Analysis of the Rabbit Sera

[0407] Plasma Lipids. The total plasma cholesterol, plasma triglyceridesand plasma phospholipids is determined enzymatically using commerciallyavailable assays according to the manufacturer's protocols (BoehringerMannheim, Mannheim, Germany and Biomérieux, 69280, Marcy-l'étoile,France).

[0408] LipoDrotein Profiles. The plasma lipoprotein profiles of thefractions obtained after the separation of the plasma into itslipoprotein fractions is determined by spinning in a sucrose densitygradient. The fractions are collected and in each individual fractionthe phospholipid and cholesterol content is measured enzymatically.

12. EXAMPLE Preparation of Peptide-Lipid Complex by Co-LyophilizationApproach

[0409] The following protocol was utilized to prepare peptide-lipidcomplexes.

[0410] One mg of peptide 210 (SEQ ID NO:210) was dissolved in 250 μlHPLC grade methanol (Perkin Elmer) in a one ml clear glass vial with cap(Waters #WAT025054). Dissolving of the peptide was aided by occasionalvortexing over a period of 10 minutes at room temperature. To thismixture an aliquot containing 3 mg dipalmitoyl-phosphatidylcholine(DPPC; Avanti Polar Lipids, 99% Purity, product #850355) from a 100mg/ml stock solution in methanol was added. The volume of the mixturewas brought to 400 μl by addition of methanol, and the mixture wasfurther vortexed intermittently for a period of 10 minutes at roomtemperature. To the tube 200 μl of xylene (Sigma-Aldrich 99% pure,HPLC-grade) was added and the tubes were vortexed for 10 seconds. Twosmall holes were punched into the top of the tube with a 20 gaugesyringe needle, the tube was frozen for 15 seconds in liquid nitrogen,and the tube was lyophilized overnight under vacuum. To the tube 200 mlsof 0.9% NaCl solution was added. The tube was vortexed for 20 seconds.At this time the solution in the tube was milky in appearance. The tubewas then incubated in a water bath for 30 minutes at 41° C. The solutionbecame clear (i.e., similar to water in appearance) after a few minutesof incubation at 41° C.

12.1. Characterizaqtion of Complexes by Superose 6 Gel FiltrationChromatography

[0411] Peptide-phospholipid complexes containing peptide 210 (SEQ IDNO:210) were prepared by colyophilization as described above. Thepreparation contained 1 mg peptide and 3 mg DPPC by weight. Afterreconstituting the complexes in 200 μl of 0.9% NaCl, 20 μl (containing100 μg peptide 210) of the complexes were applied to a PharmaciaSuperose 6 column using 0.9% NaCl as the liquid phase at a flow rate of0.5 ml/minute. The chromatography was monitored by absorbance orscattering of light of wavelength 280 nm. One ml fractions werecollected. Aliquots containing 20 μl of the fractions were assayed forphospholipid content using the bioMerieux Phospholipides Enzymatique PAP150 kit (#61491) according to the instructions supplied by themanufacturer. The vast majority of both phospholipid and UV absorbancewere recovered together in a few fractions with peaks at approximately15.1 mls. This elution volume corresponds to a Stokes' diameter of 106Angstroms.

[0412] For comparison, a separate chromatogram of 20 μl of human HDL₂was run under the same conditions and using the same column as thepeptide 210 (SEQ ID NO:210) complexes. The HDL₂ was prepared as follows:300 mls frozen human plasma (Mannheim Blutspendzentrale #1185190) wasthawed, adjusted to density 1.25 with solid potassium bromide, andcentrifuged 45 hours at 40,000 RPM using a Ti45 rotor (Beckman) at 20°C. The floating layer was collected, dialyzed against distilled water,adjusted to density 1.07 with solid potassium bromide, and centrifugedas described above for 70 hours. The bottom layer (at a level of one cmabove the tube bottom) was collected, brought to 0.01% sodium azide, andstored at 4° C. for 4 days until chromatography. The column eluate wasmonitored by absorbance or scattering of light of wavelength 254 nm. Aseries of proteins of known molecular weight and Stokes' diameter wereused as standards to calibrate the column for the calculation of Stokes'diameters of the particles (Pharmacia Gel Filtration Calibration KitInstruction Manual, Pharmacia Laboratory Separation, Piscataway, N.J.,revised April, 1985). The HDL₂ eluted with a retention volume of 14.8mls, corresponding to a Stokes' diameter of 108 nm.

13. EXAMPLE Preparation of Antibodies

[0413] To prepare antibodies to the ApoA-I agonists of the invention,peptide is conjugated to keyhole limpet hemocyanine (KLH; 1 mg peptideto 10 mg KLH). The KLH conjugate (LMG) is suspended in complete Freund'sadjuvant and injected into rabbits at time 0, and boosted with 0.25 mgKLH conjugate at 4 weeks and again at 5 weeks. Pre-bleeds and six weekpost-bleeds are tested for antibody titer against authentic antigen byELISA.

[0414] The production bleeds are pooled from 2 rabbits each. Antibodiesdirected exclusively against the peptide antigens are isolated asfollows:

[0415] 1. Free peptide is attached to cyanogen bromide activatedSepharose 4B (Pharmacia) according to the manufacturer's protocol.

[0416] 2. The antisera is preabsorbed on a column of irrelevant peptidesand on columns of irrelevant human and mouse serum proteins.

[0417] 3. The pre-absorbed antisera is passed through the correspondingpeptide column (see point 1).

[0418] 4. The columns are washed with 0.1 M borate buffered saline (pH8.2) and the bound antibodies are eluted using a low pH gradient stepfrom pH 4.0 to pH 3.0 to pH 2.0 (0.1 M glycine buffer) and finally with0.1 M HCl.

[0419] 5. The eluted material is neutralized with excess borate saline,concentrated by ultrafiltration (Amicon, YM30) and dialyzed againstborate saline.

[0420] 6. The protein concentration is determined by absorbance at 280nm.

[0421] The resulting antibodies are tested for species specificity usingpurified human ApoA-I or purified mouse ApoA-I in a direct ELISA bindingassay.

[0422] The invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

[0423] All references cited herein are incorporated herein by referencefor all purposes.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:258 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 16 (D) OTHER INFORMATION: Xaa = Naphthylalanine (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 1: Pro Val Leu Asp Leu Phe Arg Glu LeuLeu Asn Glu Leu Leu Glu Xaa 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 2: Gly Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu GluAla 1 5 10 15 Leu Lys Gln Lys Leu Lys Lys 20 (2) INFORMATION FOR SEQ IDNO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Pro Val LeuAsp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Trp 1 5 10 15 Leu LysGln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 4: Pro Val Leu Asp Leu Phe Arg Glu LeuLeu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa = D-Pro (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 5: Xaa Val Leu Asp Leu Phe Arg Glu Leu Leu AsnGlu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys Lys 20 (2)INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 6: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu AsnGlu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ProVal Leu Asp Leu Phe Lys Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 8: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Pro Val Leu Asp Leu PheArg Glu Leu Leu Asn Glu Gly Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 9: Pro Val Leu Asp Leu Phe Arg Glu Leu Gly Asn Glu Leu LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 17(D) OTHER INFORMATION: Xaa = Naphthylalanine (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 10: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu LeuGlu Ala 1 5 10 15 Xaa Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Pro ValLeu Asp Leu Phe Lys Glu Leu Leu Gln Glu Leu Leu Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 12: Pro Val Leu Asp Leu Phe Arg Glu LeuLeu Asn Glu Leu Leu Glu Ala 1 5 10 15 Gly Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 13: Gly Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Gly Leu GluAla 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 18 (D) OTHERINFORMATION: Xaa = Orn (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Pro ValLeu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 LeuXaa Gln Xaa Leu Xaa 20 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 15: Pro Val Leu Asp Leu Phe Arg Glu LeuTrp Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 16: Pro Val Leu Asp Leu Leu Arg Glu Leu Leu Asn Glu Leu Leu GluAla 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Pro Val Leu Glu LeuPhe Lys Glu Leu Leu Gln Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln LysLeu Lys 20 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 18: Gly Val Leu Asp Leu Phe Arg Glu LeuLeu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa = D-Pro (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 19: Xaa Val Leu Asp Leu Phe Arg Glu Leu Leu AsnGlu Gly Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: ProVal Leu Asp Leu Phe Arg Glu Gly Leu Asn Glu Leu Leu Glu Ala 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 21: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1 (D) OTHERINFORMATION: Xaa = D-Pro (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: XaaVal Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 22: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Pro Val Leu Asp Leu PheArg Glu Leu Leu Asn Glu Leu Leu Glu Gly 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 23: Pro Leu Leu Glu Leu Phe Lys Glu Leu Leu Gln Glu Leu LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Pro ValLeu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 LeuGln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: Pro Val Leu Asp Phe PheArg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 26: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu LeuGlu Leu 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 14(D) OTHER INFORMATION: Xaa = Naphthylalanine (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 27: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu XaaGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Pro ValLeu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Trp Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 29: Ala Val Leu Asp Leu Phe Arg Glu LeuLeu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1...22 (D) OTHER INFORMATION: N-terminal dansylatedpeptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Pro Val Leu Asp LeuPhe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln LysLeu Lys 20 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Pro Val Leu Asp Leu PheLeu Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 1 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 32: Xaa Val Leu Asp Leu Phe Arg Glu LeuLeu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 33: Pro Val Leu Asp Leu Phe Arg Glu Lys Leu Asn Glu Leu Leu GluAla 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 5 (D) OTHERINFORMATION: Xaa = Naphthylalanine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Pro Val Leu Asp Xaa Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 510 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 35: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: Pro Val Leu Asp Trp PheArg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 36: Pro Leu Leu Glu Leu Leu Lys Glu Leu Leu Gln Glu Leu LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: Pro ValLeu Asp Leu Phe Arg Glu Trp Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Pro Val Leu Asp Leu PheArg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Trp Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 39: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Glu Glu Leu LeuLys Ala 1 5 10 15 Leu Lys Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: Pro ValLeu Asp Leu Phe Asn Glu Leu Leu Arg Glu Leu Leu Glu Ala 1 5 10 15 LeuGln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Pro Val Leu Asp Leu TrpArg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 42: Pro Val Leu Asp Glu Phe Arg Glu LysLeu Asn Glu Xaa Trp Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 43: Pro Val Leu Asp Glu Phe Arg Glu Lys Leu TrpGlu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1...22 (D) OTHER INFORMATION: All genetically encoded amino acids are inthe D-configuration (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: Pro ValLeu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: Pro Val Leu Asp Glu PheArg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 46: Pro Val Leu Asp Leu Phe Arg Glu LysLeu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 47: Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Gly Leu Glu AlaLeu 1 5 10 15 Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 48:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1 (D) OTHERINFORMATION: Xaa = D-Pro (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: XaaXaa Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 49: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: Pro Val Leu Asp Leu PheArg Asn Leu Leu Glu Lys Leu Leu Glu Ala 1 5 10 15 Leu Glu Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 50: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 50: Pro Val Leu Asp Leu Phe Arg Glu LeuLeu Trp Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 51: Pro Val Leu Asp Leu Phe Trp Glu Leu Leu AsnGlu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 52: Pro Val Trp Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 53:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: Val Val Leu Asp Leu PheArg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 54: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 54: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Trp LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: Pro LeuPhe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala Leu Lys Gln 1 5 10 15 LysLeu Lys (2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 56: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Lys Lys 20 (2) INFORMATION FOR SEQ IDNO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: Pro ValLeu Asp Leu Phe Arg Asn Leu Leu Glu Glu Leu Leu Lys Ala 1 5 10 15 LeuGlu Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: Pro Val Leu Asp Glu PheArg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu 20(2) INFORMATION FOR SEQ ID NO: 59: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 59: Leu Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu GluAla 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: Pro Val Leu Asp LeuPhe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln (2)INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 61: Pro Val Leu Asp Glu Phe Arg Trp Lys Leu AsnGlu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 62: Pro Val Leu Asp Glu Trp Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 63:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: Pro ValLeu Asp Phe Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 64: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: Pro Trp Leu Asp Glu PheArg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 65: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 12 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 65: Val Leu Asp Glu Phe Arg Glu Lys LeuAsn Glu Xaa Leu Glu Ala Leu 1 5 10 15 Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 66: Pro Val Leu Asp Leu Phe Arg Asn Leu Leu Glu Glu Leu Leu GluAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: Val Leu Asp Leu PheArg Glu Leu Leu Asn Glu Leu Leu Glu Ala Leu 1 5 10 15 Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 68: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 68: Pro Val Leu Asp Glu Phe Arg Glu LeuLeu Lys Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 69: Pro Val Leu Asp Glu Phe Arg Lys Lys Leu AsnGlu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 70: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 70: Pro Val Leu Asp Glu Phe Arg Glu Leu Leu Tyr Glu Xaa Leu Glu Ala1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 71:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 14 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: Pro ValLeu Asp Glu Phe Arg Glu Lys Leu Asn Glu Leu Xaa Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 72: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: Pro Val Leu Asp Leu PheArg Glu Leu Leu Asn Glu Xaa Leu Trp Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 73: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 73: Pro Val Leu Asp Glu Phe Trp Glu LysLeu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 74: Pro Val Leu Asp Lys Phe Arg Glu Lys Leu AsnGlu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 75: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: ProVal Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Glu Leu Glu Ala 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 76: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: Pro ValLeu Asp Glu Phe Arg Glu Leu Leu Phe Glu Xaa Leu Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: Pro Val Leu Asp Glu PheArg Glu Lys Leu Asn Lys Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 78: Pro Val Leu Asp Glu Phe Arg Asp LysLeu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 79: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 79: Pro Val Leu Asp Glu Phe Arg Glu Leu Leu Asn Glu Leu Leu GluAla 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:80: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: Pro Val Leu Asp LeuPhe Glu Arg Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Gln Lys LysLeu Lys 20 (2) INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: Pro Val Leu Asp Glu PheArg Glu Lys Leu Asn Trp Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 82: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 11 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 82: Leu Asp Glu Phe Arg Glu Lys Leu AsnGlu Xaa Leu Glu Ala Leu Lys 1 5 10 15 Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 83: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 83: Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala1 5 10 15 Leu Trp Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 84:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: Pro Val Leu Asp Glu PheArg Glu Lys Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 85: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 85: Pro Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu GluAla Leu 1 5 10 15 Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:86: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: Pro Val Leu Glu LeuPhe Glu Arg Leu Leu Asp Glu Leu Leu Asn Ala 1 5 10 15 Leu Gln Lys LysLeu Lys 20 (2) INFORMATION FOR SEQ ID NO: 87: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...22 (D) OTHER INFORMATION:All amino acids are in the D- configuration (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 87: Pro Leu Leu Glu Leu Leu Lys Glu Leu Leu Gln Glu Leu LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 88: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13(D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: Pro Val Leu Asp Lys Phe Arg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 510 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 89: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: Pro ValLeu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Trp Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 90: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 10 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: Asp Glu Phe Arg Glu LysLeu Asn Glu Xaa Leu Glu Ala Leu Lys Gln 1 5 10 15 Lys Leu Lys (2)INFORMATION FOR SEQ ID NO: 91: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 91: Pro Val Leu Asp Glu Phe Arg Glu Leu Leu AsnGlu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 92: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 92: Pro Val Leu Asp Glu Phe Arg Glu Leu Tyr Asn Glu Xaa Leu Glu Ala1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 93:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: Pro ValLeu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Lys Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 94: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 94: Pro Val Leu Asp Glu Phe Arg Glu LysLeu Asn Glu Ala Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 95: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 95: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu AsnLeu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 96: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1...22 (D) OTHER INFORMATION: All genetically encoded amino acids are inthe D-configuration (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96: Pro ValLeu Asp Leu Phe Arg Glu Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 97: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 97: Pro Val Leu Asp Leu Phe Arg Glu LeuLeu Asn Glu Leu Leu Glu 1 5 10 15 FORMATION FOR SEQ ID NO: 98: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: Pro Val Leu Asp Leu PheArg Glu Leu Leu Asn Glu Glu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 99: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 99: Lys Leu Lys Gln Lys Leu Ala Glu Leu Leu Glu Asn Leu LeuGlu Arg 1 5 10 15 Phe Leu Asp Leu Val Pro 20 (2) INFORMATION FOR SEQ IDNO: 100: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1...22 (D) OTHER INFORMATION: All amino acids are in the D-configuration (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: Pro Val Leu AspLeu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys GlnLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 101: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101: Pro Val Leu Asp Leu PheArg Glu Leu Leu Asn Trp Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 102: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 102: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Asn Leu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Glu Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 103: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 103: Pro Val Leu Asp Glu Phe Arg Glu Leu Leu Asn Glu Glu Leu GluAla 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:104: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104: Pro Leu Leu AsnGlu Leu Leu Glu Ala Leu Lys Gln Lys Leu Lys 1 5 10 15 FORMATION FOR SEQID NO: 105: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: Pro AlaAla Asp Ala Phe Arg Glu Ala Ala Asn Glu Ala Ala Glu Ala 1 5 10 15 AlaLys Gln Lys Ala Lys 20 (2) INFORMATION FOR SEQ ID NO: 106: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 106: Pro Val Leu Asp Leu Phe Arg GluLys Leu Asn Glu Glu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 107: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1...22 (D) OTHER INFORMATION: All amino acids are inthe D- configuration (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107: Lys LeuLys Gln Lys Leu Ala Glu Leu Leu Glu Asn Leu Leu Glu Arg 1 5 10 15 PheLeu Asp Leu Val Pro 20 (2) INFORMATION FOR SEQ ID NO: 108: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa= Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: Pro Val Leu Asp Leu PheArg Trp Leu Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 109: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 109: Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Arg LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 110: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13(D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Xaa Glu Ala 1 510 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 111: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: ProVal Leu Asp Glu Phe Arg Glu Lys Leu Trp Glu Xaa Trp Glu Ala 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 112: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: ProVal Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Ser Glu Ala 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 113: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: Pro Val Leu Asp Glu PheArg Glu Lys Leu Asn Glu Pro Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 114: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 114: Pro Val Leu Asp Glu Phe Arg GluLys Leu Asn Glu Xaa Met Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 115: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 115: Pro Lys Leu Asp Glu Phe Arg Glu Lys Leu AsnGlu Xaa Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 116: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:13 (D) OTHER INFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 116: Pro His Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 117:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: ProGlu Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 118: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 13 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: ProVal Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Leu Glu Ala 1 5 10 15Leu Glu Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 119: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 17 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: ProVal Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Glu Leu Glu Ala 1 5 10 15Xaa Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 120: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 16 (D) OTHERINFORMATION: Xaa = Aib (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120: ProVal Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Glu Leu Glu Xaa 1 5 10 15Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 121: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: Pro Val Leu Asp Glu PheArg Glu Lys Leu Asn Glu Glu Leu Glu Ala 1 5 10 15 Leu Trp Gln Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 122: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 122: Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Glu LeuGlu Trp 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 123: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123: Gln ValLeu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 124: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 18 (D) OTHER INFORMATION: Xaa= Orn (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124: Pro Val Leu Asp Leu PheXaa Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Xaa Gln Xaa LeuXaa 20 (2) INFORMATION FOR SEQ ID NO: 125: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 125: Asn Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 126: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126: Pro ValLeu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Gly Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 127: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 127: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Asn Glu Leu Leu Glu Leu 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 128: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 128: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu GluPhe 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:129: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129: Pro Val Leu GluLeu Phe Asn Asp Leu Leu Arg Glu Leu Leu Glu Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 130: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 130: Pro Val Leu Glu Leu Phe Asn AspLeu Leu Arg Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 131: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 131: Pro Val Leu Glu Leu Phe Lys Glu Leu Leu Asn Glu Leu Leu AspAla 1 5 10 15 Leu Arg Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:132: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132: Pro Val Leu AspLeu Phe Arg Glu Leu Leu Glu Asn Leu Leu Glu Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 133: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 133: Pro Val Leu Glu Leu Phe Glu ArgLeu Leu Glu Asp Leu Leu Gln Ala 1 5 10 15 Leu Asn Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 134: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 134: Pro Val Leu Glu Leu Phe Glu Arg Leu Leu Glu Asp Leu Leu LysAla 1 5 10 15 Leu Asn Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:135: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135: Asp Val Leu AspLeu Phe Arg Glu Leu Leu Asn Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys GlnLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 136: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 136: Pro Ala Leu Glu Leu Phe Lys AspLeu Leu Gln Glu Leu Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 137: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 17 (D) OTHER INFORMATION: Xaa = Naphthylalanine (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 137: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Asn Glu Gly Leu Glu Ala 1 5 10 15 Xaa Lys Gln Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 138: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 138: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Gly Leu GluTrp 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:139: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139: Pro Val Leu AspLeu Phe Arg Glu Leu Trp Asn Glu Gly Leu Glu Ala 1 5 10 15 Leu Lys GlnLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 140: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 18 (D) OTHER INFORMATION: Xaa= Orn (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: Pro Val Leu Asp Leu PheArg Glu Leu Leu Asn Glu Gly Leu Glu Ala 1 5 10 15 Leu Xaa Gln Xaa LeuXaa 20 (2) INFORMATION FOR SEQ ID NO: 141: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 141: Pro Val Leu Asp Phe Phe Arg Glu Leu Leu Asn Glu Gly LeuGlu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 142: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142: Pro ValLeu Glu Leu Phe Arg Glu Leu Leu Asn Glu Gly Leu Glu Ala 1 5 10 15 LeuLys Gln Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 143: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...22 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 143: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu AsnGlu Gly Leu Glu Ala 1 5 10 15 Leu Lys Gln Lys Leu Lys 20 (2) INFORMATIONFOR SEQ ID NO: 144: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1 (D) OTHER INFORMATION: Xaa = D-Pro (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 144: Xaa Val Leu Glu Leu Phe Glu Asn Leu Leu Glu Arg Leu Leu Asp Ala1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 145:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145: Gly Val Leu Glu Leu PheGlu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln Lys Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 146: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 146: Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Glu Arg Leu LeuAsp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 147: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147: Pro ValLeu Glu Leu Phe Glu Asn Leu Leu Glu Arg Leu Phe Asp Ala 1 5 10 15 LeuGln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 148: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 148: Pro Val Leu Glu Leu Phe Glu AsnLeu Leu Glu Arg Leu Gly Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 149: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 149: Pro Val Leu Glu Leu Phe Glu Asn Leu Trp Glu Arg Leu Leu AspAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:150: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150: Pro Leu Leu GluLeu Phe Glu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 151: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 151: Pro Val Leu Glu Leu Phe Glu AsnLeu Gly Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 152: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 152: Pro Val Phe Glu Leu Phe Glu Asn Leu Leu Glu Arg Leu Leu AspAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:153: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153: Ala Val Leu GluLeu Phe Glu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 154: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 154: Pro Val Leu Glu Leu Phe Glu AsnLeu Leu Glu Arg Gly Leu Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 155: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 155: Pro Val Leu Glu Leu Phe Leu Asn Leu Trp Glu Arg Leu Leu AspAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:156: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156: Pro Val Leu GluLeu Phe Leu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 157: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 157: Pro Val Leu Glu Phe Phe Glu AsnLeu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 158: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 158: Pro Val Leu Glu Leu Phe Leu Asn Leu Leu Glu Arg Leu Leu AspTrp 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:159: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159: Pro Val Leu AspLeu Phe Glu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 160: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 160: Pro Val Leu Glu Leu Phe Glu AsnLeu Leu Glu Arg Leu Leu Asp Trp 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 161: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 161: Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Glu Arg Leu Leu GluAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:162: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162: Pro Val Leu GluLeu Phe Glu Asn Trp Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 163: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 163: Pro Val Leu Glu Leu Phe Glu AsnLeu Leu Glu Arg Leu Trp Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 164: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 164: Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Glu Arg Leu Leu AspAla 1 5 10 15 Trp Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:165: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165: Pro Val Leu GluLeu Phe Glu Asn Leu Leu Glu Arg Leu Leu Asp Leu 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 166: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 166: Pro Val Leu Glu Leu Phe Leu AsnLeu Leu Glu Lys Leu Leu Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 167: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 167: Pro Val Leu Glu Leu Phe Glu Asn Gly Leu Glu Arg Leu Leu AspAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:168: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168: Pro Val Leu GluLeu Phe Glu Gln Leu Leu Glu Lys Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 169: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 169: Pro Val Leu Glu Leu Phe Glu AsnLeu Leu Glu Lys Leu Leu Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 170: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 12 (D) OTHER INFORMATION: Xaa = Orn (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 170: Pro Val Leu Glu Leu Phe Glu Asn Leu Leu GluXaa Leu Leu Asp Ala 1 5 10 15 Leu Gln Xaa Xaa Leu Xaa 20 (2) INFORMATIONFOR SEQ ID NO: 171: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171: ProVal Leu Glu Leu Phe Glu Asn Leu Leu Glu Lys Leu Leu Asp Leu 1 5 10 15Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 172: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172: Pro Val Leu Glu Leu PheLeu Asn Leu Leu Glu Arg Leu Gly Asp Ala 1 5 10 15 Leu Gln Lys Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 173: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 173: Pro Val Leu Asp Leu Phe Asp Asn Leu Leu Asp Arg Leu LeuAsp Leu 1 5 10 15 Leu Asn Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 174: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1...22 (D) OTHER INFORMATION: All amino acids are in the D-configuration (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 174: Pro Val Leu GluLeu Phe Glu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 175: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 175: Pro Val Leu Glu Leu Phe Glu AsnLeu Leu Glu Arg Leu Leu Glu Leu 1 5 10 15 Leu Asn Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 176: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 176: Pro Val Leu Glu Leu Trp Glu Asn Leu Leu Glu Arg Leu Leu AspAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:177: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 177: Gly Val Leu GluLeu Phe Leu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 178: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 178: Pro Val Leu Glu Leu Phe Asp AsnLeu Leu Glu Lys Leu Leu Glu Ala 1 5 10 15 Leu Gln Lys Lys Leu Arg 20 (2)INFORMATION FOR SEQ ID NO: 179: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 179: Pro Val Leu Glu Leu Phe Asp Asn Leu Leu Glu Arg Leu Leu AspAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:180: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 180: Pro Val Leu GluLeu Phe Asp Asn Leu Leu Asp Lys Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Arg 20 (2) INFORMATION FOR SEQ ID NO: 181: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 181: Pro Val Leu Glu Leu Phe Glu AsnLeu Leu Glu Arg Trp Leu Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 182: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 182: Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Glu Lys Leu Leu GluAla 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO:183: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 183: Pro Leu Leu GluLeu Phe Glu Asn Leu Leu Glu Lys Leu Leu Asp Ala 1 5 10 15 Leu Gln LysLys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 184: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 184: Pro Val Leu Glu Leu Phe Leu AsnLeu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Trp Gln Lys Lys Leu Lys 20 (2)INFORMATION FOR SEQ ID NO: 185: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 19 (D) OTHER INFORMATION: Xaa = Orn (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 185: Pro Val Leu Glu Leu Phe Glu Asn Leu Leu GluArg Leu Leu Asp Ala 1 5 10 15 Leu Gln Xaa Xaa Leu Xaa 20 (2) INFORMATIONFOR SEQ ID NO: 186: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 186: ProVal Leu Glu Leu Phe Glu Gln Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 187: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 187: Pro Val Leu Glu Leu PheGlu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 Leu Asn Lys Lys LeuLys 20 (2) INFORMATION FOR SEQ ID NO: 188: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 188: Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Asp Arg Leu LeuAsp Ala 1 5 10 15 Leu Gln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ IDNO: 189: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 189: Asp ValLeu Glu Leu Phe Glu Asn Leu Leu Glu Arg Leu Leu Asp Ala 1 5 10 15 LeuGln Lys Lys Leu Lys 20 (2) INFORMATION FOR SEQ ID NO: 190: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 190: Pro Val Leu Glu Phe Trp Asp AsnLeu Leu Asp Lys Leu Leu Asp Ala 1 5 10 15 Leu Gln Lys Lys Leu Arg 20 (2)INFORMATION FOR SEQ ID NO: 191: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1...18 (D) OTHER INFORMATION: N-terminal acetylatedand C-terminal amidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 191: ProVal Leu Asp Leu Leu Arg Glu Leu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15Leu Lys (2) INFORMATION FOR SEQ ID NO: 192: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 192: Pro Val Leu Asp Leu Phe Lys Glu Leu Leu Glu Glu Leu LysGln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 193: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 193: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu GluGlu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO:194: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 194: Pro Val Leu Glu Leu Phe Arg GluLeu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FORSEQ ID NO: 195: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1...18 (D) OTHER INFORMATION: N-terminal acetylated and C-terminalamidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 195: Pro Val Leu Glu LeuPhe Lys Glu Leu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2)INFORMATION FOR SEQ ID NO: 196: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1...18 (D) OTHER INFORMATION: N-terminal acetylatedand C-terminal amidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 196: ProVal Leu Asp Leu Phe Arg Glu Leu Leu Glu Glu Leu Lys Asn Lys 1 5 10 15Leu Lys (2) INFORMATION FOR SEQ ID NO: 197: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 197: Pro Leu Leu Asp Leu Phe Arg Glu Leu Leu Glu Glu Leu LysGln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 198: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 198: Gly Val Leu Asp Leu Phe Arg Glu Leu Leu GluGlu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO:199: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 199: Pro Val Leu Asp Leu Phe Arg GluLeu Trp Glu Glu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FORSEQ ID NO: 200: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1...18 (D) OTHER INFORMATION: N-terminal acetylated and C-terminalamidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 200: Asn Val Leu Asp LeuPhe Arg Glu Leu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2)INFORMATION FOR SEQ ID NO: 201: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1...18 (D) OTHER INFORMATION: N-terminal acetylatedand C-terminal amidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 201: ProLeu Leu Asp Leu Phe Lys Glu Leu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15Leu Lys (2) INFORMATION FOR SEQ ID NO: 202: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 202: Pro Ala Leu Glu Leu Phe Lys Asp Leu Leu Glu Glu Leu ArgGln Lys 1 5 10 15 Leu Arg (2) INFORMATION FOR SEQ ID NO: 203: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 203: Ala Val Leu Asp Leu Phe Arg Glu Leu Leu GluGlu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO:204: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 204: Pro Val Leu Asp Phe Phe Arg GluLeu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FORSEQ ID NO: 205: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1...18 (D) OTHER INFORMATION: N-terminal acetylated and C-terminalamidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 205: Pro Val Leu Asp LeuPhe Arg Glu Trp Leu Glu Glu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2)INFORMATION FOR SEQ ID NO: 206: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1...18 (D) OTHER INFORMATION: N-terminal acetylatedand C-terminal amidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 206: ProLeu Leu Glu Leu Leu Lys Glu Leu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15Leu Lys (2) INFORMATION FOR SEQ ID NO: 207: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 207: Pro Val Leu Glu Leu Leu Lys Glu Leu Leu Glu Glu Leu LysGln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 208: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 208: Pro Ala Leu Glu Leu Phe Lys Asp Leu Leu GluGlu Leu Arg Gln Arg 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO:209: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 209: Pro Val Leu AspLeu Phe Arg Glu Leu Leu Asn Glu Leu Leu Gln Lys 1 5 10 15 Leu Lys (2)INFORMATION FOR SEQ ID NO: 210: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 210: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Glu Glu Leu Lys GlnLys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 211: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 211: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Glu Glu Leu XaaGln Xaa 1 5 10 15 Leu Xaa (2) INFORMATION FOR SEQ ID NO: 212: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 212: Pro Val Leu Asp Leu Phe Xaa Glu Leu Leu GluGlu Leu Xaa Gln Xaa 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO:213: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 213: Pro Ala Leu Glu Leu Phe Lys AspLeu Leu Glu Glu Phe Arg Gln Arg 1 5 10 15 Leu Lys (2) INFORMATION FORSEQ ID NO: 214: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:1...18 (D) OTHER INFORMATION: N-terminal acetylated and C-terminalamidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 214: Pro Val Leu Asp LeuPhe Arg Glu Leu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2)INFORMATION FOR SEQ ID NO: 215: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1...18 (D) OTHER INFORMATION: N-terminal acetylatedand C-terminal amidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 215: ProVal Leu Asp Leu Phe Arg Glu Leu Leu Glu Glu Trp Lys Gln Lys 1 5 10 15Leu Lys (2) INFORMATION FOR SEQ ID NO: 216: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 216: Pro Val Leu Glu Leu Phe Lys GluLeu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FORSEQ ID NO: 217: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 217: Pro ValLeu Asp Leu Phe Arg Glu Leu Leu Glu Leu Leu Lys Gln Lys 1 5 10 15 LeuLys (2) INFORMATION FOR SEQ ID NO: 218: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION: N-terminalacetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO:218: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Glu Leu Leu Gln Lys 1 510 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 219: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 219: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Asn Glu Leu Trp Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FORSEQ ID NO: 220: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 220: Pro ValLeu Asp Leu Phe Arg Glu Leu Leu Glu Glu Leu Gln Lys Lys 1 5 10 15 LeuLys (2) INFORMATION FOR SEQ ID NO: 221: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION: N-terminalacetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221: Asp Val Leu Asp Leu Phe Arg Glu Leu Leu Glu Glu Leu Lys Gln Lys 1 510 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 222: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 222: Pro Val Leu Asp Ala Phe Arg GluLeu Leu Glu Ala Leu Leu Gln Leu 1 5 10 15 Lys Lys (2) INFORMATION FORSEQ ID NO: 223: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 223: Pro ValLeu Asp Ala Phe Arg Glu Leu Leu Glu Ala Leu Ala Gln Leu 1 5 10 15 LysLys (2) INFORMATION FOR SEQ ID NO: 224: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 224: Pro Val Leu Asp Leu Phe Arg Glu Gly Trp Glu Glu Leu LysGln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 225: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 225: Pro Val Leu Asp Ala PheArg Glu Leu Ala Glu Ala Leu Ala Gln Leu 1 5 10 15 Lys Lys (2)INFORMATION FOR SEQ ID NO: 226: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 226: Pro Val Leu Asp Ala Phe Arg Glu Leu Gly Glu Ala Leu Leu GlnLeu 1 5 10 15 Lys Lys (2) INFORMATION FOR SEQ ID NO: 227: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 227: Pro Val Leu Asp Leu Phe Arg Glu Leu Gly Glu Glu Leu LysGln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 228: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 228: Pro Val Leu Asp Leu Phe Arg Glu Gly Leu GluGlu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO:229: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 229: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Glu Glu Gly Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FORSEQ ID NO: 230: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 230: Pro ValLeu Glu Leu Phe Glu Arg Leu Leu Glu Asp Leu Gln Lys Lys 1 5 10 15 LeuLys (2) INFORMATION FOR SEQ ID NO: 231: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 231: Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Glu Lys Leu GluGln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO: 232: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 232: Pro Leu Leu Glu Leu Phe Lys Glu Leu Leu GluGlu Leu Lys Gln Lys 1 5 10 15 Leu Lys (2) INFORMATION FOR SEQ ID NO:233: (i) SEQUENCE CHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:233: This sequence is intentionally skipped (2) INFORMATION FOR SEQ IDNO: 234: (i) SEQUENCE CHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 234: This sequence is intentionally skipped (2) INFORMATION FOR SEQID NO: 235: (i) SEQUENCE CHARACTERISTICS: (xi) SEQUENCE DESCRIPTION: SEQID NO: 235: This sequence is intentionally skipped (2) INFORMATION FORSEQ ID NO: 236: (i) SEQUENCE CHARACTERISTICS: (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 236: This sequence is intentionally skipped (2) INFORMATIONFOR SEQ ID NO: 237: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 aminoacids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 237: LeuAsp Asp Leu Leu Gln Lys Trp Ala Glu Ala Phe Asn Gln Leu Leu 1 5 10 15Lys Lys (2) INFORMATION FOR SEQ ID NO: 238: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 238: Glu Trp Leu Lys Ala Phe Tyr Glu Lys Val Leu Glu Lys LeuLys Glu 1 5 10 15 Leu Phe (2) INFORMATION FOR SEQ ID NO: 239: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 239: Glu Trp Leu Glu Ala Phe Tyr Lys Lys Val LeuGlu Lys Leu Lys Glu 1 5 10 15 Leu Phe (2) INFORMATION FOR SEQ ID NO:240: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 240: Asp Trp Leu Lys Ala Phe Tyr AspLys Val Ala Glu Lys Leu Lys Glu 1 5 10 15 Ala Phe (2) INFORMATION FORSEQ ID NO: 241: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 241: Asp TrpPhe Lys Ala Phe Tyr Asp Lys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 PhePhe (2) INFORMATION FOR SEQ ID NO: 242: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 242: Gly Ile Lys Lys Phe Leu Gly Ser Ile Trp Lys Phe Ile LysAla Phe 1 5 10 15 Val Gly (2) INFORMATION FOR SEQ ID NO: 243: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 243: Asp Trp Phe Lys Ala PheTyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15 Ala Phe (2)INFORMATION FOR SEQ ID NO: 244: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQID NO: 244: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu LysGlu 1 5 10 15 Ala Phe (2) INFORMATION FOR SEQ ID NO: 245: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 245: Asp Trp Leu Lys Ala Phe Tyr AspLys Val Phe Glu Lys Phe Lys Glu 1 5 10 15 Phe Phe (2) INFORMATION FORSEQ ID NO: 246: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 246: Glu TrpLeu Glu Ala Phe Tyr Lys Lys Val Leu Glu Lys Leu Lys Glu 1 5 10 15 LeuPhe (2) INFORMATION FOR SEQ ID NO: 247: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 247: Asp Trp Phe Lys Ala Phe Tyr Asp Lys Phe Phe Glu Lys PheLys Glu 1 5 10 15 Phe Phe (2) INFORMATION FOR SEQ ID NO: 248: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 248: Glu Trp Leu Lys Ala PheTyr Glu Lys Val Leu Glu Lys Leu Lys Glu 1 5 10 15 Leu Phe (2)INFORMATION FOR SEQ ID NO: 249: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix) FEATURE: (A) NAME/KEY:Other (B) LOCATION: 1...18 (D) OTHER INFORMATION: N-terminal acetylatedand C-terminal amidated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 249: GluTrp Leu Lys Ala Glu Tyr Glu Lys Val Glu Glu Lys Leu Lys Glu 1 5 10 15Leu Phe (2) INFORMATION FOR SEQ ID NO: 250: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (ix)FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHER INFORMATION:N-terminal acetylated and C-terminal amidated (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 250: Glu Trp Leu Lys Ala Glu Tyr Glu Lys Val Leu Glu Lys LeuLys Glu 1 5 10 15 Leu Phe (2) INFORMATION FOR SEQ ID NO: 251: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...18 (D) OTHERINFORMATION: N-terminal acetylated and C-terminal amidated (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 251: Glu Trp Leu Lys Ala Phe Tyr Lys Lys Val LeuGlu Lys Leu Lys Glu 1 5 10 15 Leu Phe (2) INFORMATION FOR SEQ ID NO:252: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...15 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 252: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Glu Gln Lys Leu Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:253: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...16 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 253: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Glu Glu Leu Lys Gln Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:254: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...16 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 254: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Glu Lys Leu Lys Gln Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:255: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...15 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 255: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Glu Lys Leu Gln Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:256: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...16 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 256: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Glu Ala Leu Lys Gln Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:257: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...16 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 257: Pro Val Leu Asp Leu Phe Glu AsnLeu Leu Glu Arg Leu Lys Gln Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:258: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: None (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...16 (D)OTHER INFORMATION: N-terminal acetylated and C-terminal amidated (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 258: Pro Val Leu Asp Leu Phe Arg GluLeu Leu Asn Glu Leu Lys Gln Lys 1 5 10 15

What is claimed is:
 1. An ApoA-I agonist comprising: (i) a 14 to22-residue peptide or peptide analogue which forms an amphipathicα-helix in the presence of lipids and which comprises the structuralformula (I):Z₁—X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉—X₁₀—X₁₁—X₁₂—X₁₃—X₁₄—X₁₅—X₁₆—X₁₇—X₁₈ X₁ isPro (P), Ala (A), Gly (G), Asn (N), Gln (Q) or D-Pro (p); X₂ is analiphatic amino acid; X₃ is Leu (L); X₄ is an acidic amino acid; X₅ isLeu (L) or Phe (F); X₆ is Leu (L) or Phe (F); X₇ is a basic amino acid;X₈ is an acidic amino acid; X₉ is Leu (L) or Trp (W); X₁₀ is Leu (L) orTrp (W); X₁₁ is an acidic amino acid or Asn (N); X₁₂ is an acidic aminoacid; X₁₃ is Leu (L), Trp (W) or Phe (F); X₁₄ is a basic amino acid orLeu (L); X₁₅ is Gln (Q) or Asn (N); X₁₆ is a basic amino acid; X₁₇ isLeu (L); X₁₈ is a basic amino acid; Z₁ is H₂N— or RC(O)NH—; Z₂ is—C(O)NRR, —C(O)OR or —C(O)OH or a salt thereof; each R is independently—H, (C₁-C₆) alkyl, (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, (C₅-C₂₀) aryl,(C₆-C₂6) alkaryl, 5-20 membered heteroaryl or 6-26 memberedalkheteroaryl or a 1 to 4-residue peptide or peptide analogue; each “—”between residues X_(n) independently designates an amide linkage, asubstituted amide linkage, an isostere of an amide or an amide mimetic;or (ii) a deleted from of structural formula (I) in which at least oneand up to eight of residues X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀,X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇ and X₁₈ are deleted; or (iii) analtered form of structural formula (I) in which at least one of residuesX₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆,X₁₇ or X₁₈ is conservatively substituted with another residue.
 2. TheApoA-I agonist of claim 1 which exhibits at least about 38%LCAT-activation activity as compared with human ApoA-I.
 3. The ApoA-Iagonist of claim 1 which is the altered form of structural formula (I).4. The ApoA-I agonist of claim 3 in which the hydrophobic residues arefixed according to structural formula (I) and at least one non-fixedresidue is conservatively substituted with another residue.
 5. TheApoA-I agonist of claim 4 in which: X₁ is Pro (P), D-Pro (p), Gly (G),Asn (N) or Ala (A); X₂ is Ala (A), Leu (L) or Val (V); X₃ is Leu (L); X₅is Leu (L) or Phe (F); X₆ is Leu (L) or Phe (F); X₉ is Leu (L) or Trp(W); X₁₀ is Leu (L) or Trp (W); X₁₃ is Leu (L), Trp (W) or Phe (F); X₁₇is Leu (L); and at least one of X₄, X₇, X₈, X₁₁, X₁₂, X₁₄, X₁₅, X₁₆ andX₁₈ is conservatively substituted with another residue.
 6. The ApoA-Iagonist of claim 3 in which the hydrophilic residues are fixed accordingto structural formula (I) and at least one non-fixed residue isconservatively substituted with another residue.
 7. The ApoA-I agonistof claim 6 in which: X₄ is Asp (D) or Glu (E); X₇ is Arg (R), Lys (K) orOrn; X₈ is Asp (D) or Glu (E); X₁₁ is Asn (N) or Glu (E); X₁₂ is Glu(E); X₁₄ is Lys (K), Arg (R) or Orn; X₁₅ is Gln (Q) or Asn (N); X₁₆ isLys (K), Arg (R) or Orn; X₁₈ is Asn (N) or Gln (Q); and at least one ofX₁, X₂, X₃, X₅, X₆, X₉, X₁₀, X₁₃ and X₁₇ is conservatively substitutedwith another residue.
 8. The ApoA-I agonist of claim 6 in which X₃ isLeu (L), X₆ is Phe (F), X₉ is Leu (L) or Trp (W), X₁₀ is Leu (L) or Trp(W) and at least one of X₁, X₂, X₅, X₁₃ and X₁₇ is conservativelysubstituted with another residue.
 9. The ApoA-I agonist of claim 5 or 7in which the substituting residue is classified within the samesub-category as the substituted residue.
 10. The ApoA-I agonist of claim1 which is the deleted form of structural formula (I).
 11. The ApoA-Iagonist of claim 10 in which one helical turn of the peptide or peptideanalogue is deleted.
 12. The ApoA-I agonist of claim 1 which is an18-residue peptide or peptide analogue of structural formula (I). 13.The ApoA-I agonist of claim 12 in which: the “—” between residuesdesignates —C(O)NH—; Z₁ is H₂N—; and Z₂ is —C(O)OH or a salt thereof.14. The ApoA-I agonist of claim 13, in which: X₁ is Pro (P), Ala (A),Gly (G), Asn (N) or D-Pro (p); X₂ is Ala (A), Val (V) or Leu (L); X₃ isLeu (L); X₄ is Asp (D) or Glu (E); X₅ is Leu (L) or Phe (F); X₆ is Leu(L) or Phe (F); X₇ is Arg (R), Lys (K) or Orn; X₈ is Asp (D) or Glu (E);X₉ is Leu (L) or Trp (W); X₁₀ is Leu (L) or Trp (W); X₁₁ is Glu (E) orAsn (N); X₁₂ is Glu (E); X₁₃ is Leu (L), Trp (W) or Phe (F); X₁₄ is Arg(R), Lys (K) or Orn; X₁₅ is Gln (Q) or Asn (N); X₁₆ is Arg (R), Lys (K)or Orn; X₁₇ is Leu (L); and X₁₈ is Arg (R), Lys (K) or Orn.
 15. TheApoA-I agonist of claim 1 which is selected from the group consistingof: peptide 191 PVLDLLRELLEELKQKLK* (SEQ ID NO:194); peptide 192PVLDLFKELLEELKQKLK* (SEQ ID NO:192); peptade 193 PVLDLFRELLEELKQKLK*(SEQ ID NO:193); peptide 194 PVLELFRELLEELKQKLK* (SEQ ID NO:194);peptide 195 PVLELFKELLEELKQKLK* (SEQ ID NO:195); peptide 196PVLDLFRELLEELKNKLK* (SEQ ID NO:196); peptide 197 PLLDLFRELLEELKQKLK*(SEQ ID NO:197); peptide 198 GVLDLFRELLEELKQKLK* (SEQ ID NO:198);peptide 199 PVLDLFRELWEELKQKLK* (SEQ ID NO:199); peptide 200NVLDLFRELLEELKQKLK* (SEQ ID NO:200); peptide 201 PLLDLFKELLEELKQKLK*(SEQ ID NO:201); peptide 202 PALELFKDLLEELRQKLR* (SEQ ID NO:202);peptide 203 AVLDLFRELLEELKQKLK* (SEQ ID NO:203); peptide 204PVLDFFRELLEELKQKLK* (SEQ ID NO:204); peptide 205 PVLDLFREWLEELKQKLK*(SEQ ID NO:205); peptide 206 PLLELLKELLEELKQKLK* (SEQ ID NO:206);peptide 207 PVLELLKELLEELKQKLK* (SEQ ID NO:207); peptide 208PALELFKDLLEELRQRLK* (SEQ ID NO:208); peptide 209 PVLDLFRELLNELLQKLK (SEQID NO:209); peptide 210 PVLDLFRELLEELKQKLK (SEQ ID NO:210); peptide 211PVLDLFRELLEELOQOLO* (SEQ ID NO:211); peptide 212 PVLDLFOELLEELOQOLK*(SEQ ID NO:212); peptide 213 PALELFKDLLEEFRQRLK* (SEQ ID NO:213);peptide 214 pVLDLFRELLEELKQKLK* (SEQ ID NO:214); peptide 215PVLDLFRELLEEWKQKLK* (SEQ ID NO:215); peptide 229 PVLELFERLLEDLQKKLK (SEQID NO:229); peptide 230 PVLDLFRELLEKLEQKLK (SEQ ID NO:230); peptide 231PLLELFKELLEELKQKLK* (SEQ ID NO:2324;

in either the N- and/or C-terminal blocked or unblocked forms.
 16. Amultimeric ApoA-I agonist which exhibits at least about 38% LCATactivation activity as compared with human ApoA-I and which has thestructural formula (II): HHLL_(m)—HH_(n)LL_(m)—HH  (II) or apharmaceutically acceptable salt thereof, wherein: each m isindependently an integer from 0 to 1; n is an integer from 0 to 10; each“HH” is independently a peptide or peptide analogue according to claim1; each “LL” is independently a bifunctional linker; and each “—”independently designates a covalent linkage.
 17. A multimeric ApoA-Iagonist which exhibits at least about 38% LCAT activation activity ascompared with human ApoA-I and which has the structural formula (III):X—N_(ya)—X_((ya−1))N_(yb)—X_((yb−1)))_(p)  (III) or a pharmaceuticallyacceptable salt thereof, wherein: each X is independentlyHHLL_(m)—HH_(n)LL_(m)—HH; each HH is independently a core peptide ofstructure (I) or an analogue or mutated, truncated, internally deletedor extended form thereof as described herein; each LL is independently abifunctional linker; each m is independently an integer from 0 to 1;each n is independently an integer from 0 to 8; N_(ya) and N_(yb) areeach independently a multifunctional linking moiety where y_(a) andy_(b) represent the number of functional groups on N_(ya) and N_(yb),respectively; each y_(a) or y_(b) is independently an integer from 3 to8; p is an integer from 0 to 7; and each “—” independently designates acovalent bond.
 18. A multimeric ApoA-I agonist which exhibits at leastabout 38% LCAT activation activity as compared with human ApoA-I andwhich has the structural formula (IV) or (V):

or a pharmaceutically acceptable salt thereof, wherein: each X isindependently HHLL_(m)—HH_(n)LL_(m)HH; each HH is independently apeptide or peptide analogue according to claim 1; each LL isindependently a bifunctional linker; each n is independently an integerfrom 0 to 1; each m is independently an integer from 0 to 8; R₁ is —ORor —NRR; and each R is independently —H, (C₁-C₆) alkyl, (C₁-C₆) alkenyl,(C₁-C₆) alkynyl; (C₅-C₂₀) aryl (C₆-C₂₆) alkaryl, 5-20 memberedheteroaryl or 6-26 membered alkheteroaryl.
 19. The multimeric ApoA-Iagonist of claim 16, 17 or 18 in which the bifunctional linker iscleavable.
 20. The ApoA-I multimeric agonist of claim 16, 17 or 18 inwhich n is
 0. 21. The multimeric ApoA-I agonist of claim 20 in which mis
 0. 22. The multimeric ApoA-I agonist of claim 16, 17 or 18 in whicheach HH is independently a peptide according to claim
 13. 23. Themultimeric ApoA-I agonist of claim 16, 17 or 18 in which each HH isindependently a peptide according to claim
 14. 24. The multimeric ApoA-Iagonist of claim 16, 17 or 18 in which each HH is independently apeptide according to claim
 15. 25. An ApoA-I agonist-lipid complexcomprising an ApoA-I agonist and a lipid, wherein the ApoA-I agonist isa peptide or peptide analogue according to claim 1, a multimeric ApoA-Iagonist according to claim 16, a multimeric ApoA-I agonist according toclaim 17, or a multimeric ApoA-I agonist according to claim
 18. 26. TheApoA-I agonist-lipid complex of claim 25 in which the ApoA-I agonist isa peptide according to claim
 12. 27. The ApoA-I agonist-lipid complex ofclaim 25 in which the ApoA-I agonist is a peptide according to claim 13.28. The ApoA-I agonist-lipid complex of claim 25 in which the ApoA-Iagonist is a peptide according to claim
 14. 29. The ApoA-I agonist-lipidcomplex of claim 25 in which the ApoA-I agonist is a peptide accordingto claim
 15. 30. The ApoA-I agonist-lipid complex of claim 25 in whichthe lipid is sphingomyelin.
 31. The ApoA-I agonist-lipid complex ofclaim 25 which is in the form of a lyophilized powder.
 32. The ApoA-Iagonist-lipid complex of claim 25 which is in the form of a solution.33. A pharmaceutical composition comprising an ApoA-I agonist and apharmaceutically acceptable carrier, excipient or diluent, wherein theApoA-I agonist is a peptide or peptide analogue according to claim 1, amultimeric ApoA-I agonist according to claim 16, a multimeric ApoA-Iagonist according to claim 17, or a multimeric ApoA-I agonist accordingto claim
 18. 34. The pharmaceutical composition of claim 33 in which theApoA-I agonist is a peptide according to claim
 12. 35. Thepharmaceutical composition of claim 33 in which the ApoA-I agonist is apeptide according to claim
 13. 36. The pharmaceutical composition ofclaim 33 in which the ApoA-I agonist is a peptide according to claim 14.37. The pharmaceutical composition of claim 33 in which the ApoA-Iagonist is a peptide according to claim
 15. 38. The pharmaceuticalcomposition of claim 33, 34, 35, 36 or 37, in which the ApoA-I agonistis in the form of an ApoA-I agonist-lipid complex, said complexcomprising the ApoA-I agonist and a lipid.
 39. The pharmaceuticalcomposition of claim 38 in which the ApoA-I agonist-lipid complex is inthe form of a lyophilized powder.
 40. A method of treating a subjectsuffering from a disorder associated with dyslipidemia, said methodcomprising the step of administering to the subject an effective amountof the ApoA-I agonist of claim
 1. 41. The method of claim 40 in whichthe ApoA-I agonist is in the form of a pharmaceutical composition, saidcomposition comprising the ApoA-I agonist and a pharmaceuticallyacceptable carrier, excipient or diluent.
 42. The method of claim 40 inwhich the ApoA-I agonist is in the form of an ApoA-I agonist-lipidcomplex, said complex comprising the ApoA-I agonist and a lipid.
 43. Themethod of claim 40 in which the disorder associated with dyslipidemia ishypercholesterolemia.
 44. The method of claim 40 in which the disorderassociated with dyslipidemia is cardiovascular disease.
 45. The methodof claim 40 in which the disorder associated with dyslipidemia isatherosclerosis.
 46. The method of claim 40 in which the disorderassociated with dyslipidemia is restenosis.
 47. The method of claim 40,in which the disorder associated with dyslipidemia is HDL or ApoA-Ideficiency.
 48. The method of claim 40, in which the disorder associatedwith dyslipidemia is hypertriglyceridemia.
 49. The method of claim 40,in which the disorder associated with dyslipidemia is metabolicsyndrome.
 50. A method of treating a subject suffering from septicshock, said method comprising the step of administering to the subjectan effective amount of the ApoA-I agonist of claim
 1. 51. The method ofclaim 40 or 50 in which said subject is a human.
 52. The method of claim40 or 50 in which about 0.5 mg/kg to about 100 mg/kg ApoA-I agonist isadministered to said subject.